Non-specific Lethal Research Articles

KAT8 beyond Acetylation: A Survey of Its Epigenetic Regulation, Genetic Variability, and Implications for Human Health.

Lysine acetyltransferase 8, also known as KAT8, is an enzyme involved in epigenetic regulation, primarily recognized for its ability to modulate histone acetylation. This review presents an overview of KAT8, emphasizing its biological functions, which impact many cellular processes and range from chromatin remodeling to genetic and epigenetic regulation. In many model systems, KAT8's acetylation of histone H4 lysine 16 (H4K16) is critical for chromatin structure modification, which influences gene expression, cell proliferation, differentiation, and apoptosis. Furthermore, this review summarizes the observed genetic variability within the KAT8 gene, underscoring the implications of various single nucleotide polymorphisms (SNPs) that affect its functional efficacy and are linked to diverse phenotypic outcomes, ranging from metabolic traits to neurological disorders. Advanced insights into the structural biology of KAT8 reveal its interaction with multiprotein assemblies, such as the male-specific lethal (MSL) and non-specific lethal (NSL) complexes, which regulate a wide range of transcriptional activities and developmental functions. Additionally, this review focuses on KAT8's roles in cellular homeostasis, stem cell identity, DNA damage repair, and immune response, highlighting its potential as a therapeutic target. The implications of KAT8 in health and disease, as evidenced by recent studies, affirm its importance in cellular physiology and human pathology.

Mcrs1 regulates G2/M transition and spindle assembly during mouse oocyte meiosis.

Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.

Dendritic spine and synapse pathology in chromatin modifier-associated autism spectrum disorders and intellectual disability.

Formation of dendritic spine and synapse is an essential final step of brain wiring to establish functional communication in the developing brain. Recent findings have displayed altered dendritic spine and synapse morphogenesis, plasticity, and related molecular mechanisms in animal models and post-mortem human brains of autism spectrum disorders (ASD) and intellectual disability (ID). Many genes and proteins are shown to be associated with spines and synapse development, and therefore neurodevelopmental disorders. In this review, however, particular attention will be given to chromatin modifiers such as AT-Rich Interactive Domain 1B (ARID1B), KAT8 regulatory non-specific lethal (NSL) complex subunit 1 (KANSL1), and WD Repeat Domain 5 (WDR5) which are among strong susceptibility factors for ASD and ID. Emerging evidence highlights the critical status of these chromatin remodeling molecules in dendritic spine morphogenesis and synaptic functions. Molecular and cellular insights of ARID1B, KANSL1, and WDR5 will integrate into our current knowledge in understanding and interpreting the pathogenesis of ASD and ID. Modulation of their activities or levels may be an option for potential therapeutic treatment strategies for these neurodevelopmental conditions.

Protein network analysis links the NSL complex to Parkinson's disease via mitochondrial and nuclear biology.

Whilst the majority of Parkinson's Disease (PD) cases are sporadic, much of our understanding of the pathophysiological basis of the disease can be traced back to the study of rare, monogenic forms of PD. In the past decade, the availability of genome-wide association studies (GWAS) has facilitated a shift in focus, toward identifying common risk variants conferring increased risk of developing PD across the population. A recent mitophagy screening assay of GWAS candidates has functionally implicated the non-specific lethal (NSL) complex in the regulation of PINK1-mitophagy. Here, a bioinformatics approach has been taken to investigate the proteome of the NSL complex, to unpick its relevance to PD pathogenesis. The NSL interactome has been built, using 3 online tools: PINOT, HIPPIE and MIST, to mine curated, literature-derived protein-protein interaction (PPI) data. We built (i) the 'mitochondrial' NSL interactome exploring its relevance to PD genetics and (ii) the PD-oriented NSL interactome to uncover biological pathways underpinning the NSL/PD association. In this study, we find the mitochondrial NSL interactome to be significantly enriched for the protein products of PD-associated genes, including the Mendelian PD genes LRRK2 and VPS35. In addition, we find nuclear processes to be amongst those most significantly enriched within the PD-associated NSL interactome. These findings strengthen the role of the NSL complex in sporadic and familial PD, mediated by both its mitochondrial and nuclear functions.

The Non-Specific Lethal (NSL) Histone Acetyltransferase Complex Transcriptionally Regulates Yin Yang 1-Mediated Cell Proliferation in Human Cells.

The human males absent on the first (MOF)-containing non-specific lethal (NSL) histone acetyltransferase (HAT) complex acetylates histone H4 at lysine K5, K8, and K16. This complex shares several subunits with other epigenetic regulatory enzymes, which highlights the complexity of its intracellular function. However, the effect of the NSL HAT complex on the genome and target genes in human cells is still unclear. By using a CRISPR/Cas9-mediated NSL3-knockout 293T cell line and chromatin immunoprecipitation-sequencing (ChIP-Seq) approaches, we identified more than 100 genes as NSL HAT transcriptional targets, including several transcription factors, such as Yin Yang 1 (YY1) which are mainly involved in cell proliferation, biological adhesion, and metabolic processes. We found here that the ChIP-Seq peaks of MOF and NSL3 co-localized with H4K16ac, H3K4me2, and H3K4me3 at the transcriptional start site of YY1. In addition, both the mRNA and protein expression levels of YY1 were regulated by silencing or overexpressing NSL HAT. Interestingly, the expression levels of cell division cycle 6, a downstream target gene of YY1, were regulated by MOF or NSL3. In addition, the suppressed clonogenic ability of HepG2 cells caused by siNSL3 was reversed by overexpressing YY1, suggesting the involvement of YY1 in NSL HAT functioning. Additionally, de novo motif analysis of MOF and NSL3 targets indicated that the NSL HAT complex may recognize the specific DNA-binding sites in the promoter region of target genes in order to regulate their transcription.

Methyl-lysine readers PHF20 and PHF20L1 define two distinct gene expression–regulating NSL complexes

The methyl-lysine readers plant homeodomain finger protein 20 (PHF20) and its homolog PHF20-like protein 1 (PHF20L1) are known components of the nonspecific lethal (NSL) complex that regulates gene expression through its histone acetyltransferase activity. In the current model, both PHF homologs coexist in the same NSL complex, although this was not formally tested; nor have the functions of PHF20 and PHF20L1 regarding NSL complex integrity and transcriptional regulation been investigated. Here, we perform an in-depth biochemical and functional characterization of PHF20 and PHF20L1 in the context of the NSL complex. Using mass spectrometry, genome-wide chromatin analysis, and protein-domain mapping, we identify the existence of two distinct NSL complexes that exclusively contain either PHF20 or PHF20L1. We show that the C-terminal domains of PHF20 and PHF20L1 are essential for complex formation with NSL, and the Tudor 2 domains are required for chromatin binding. The genome-wide chromatin landscape of PHF20–PHF20L1 shows that these proteins bind mostly to the same genomic regions, at promoters of highly expressed/housekeeping genes. Yet, deletion of PHF20 and PHF20L1 does not abrogate gene expression or impact the recruitment of the NSL complex to those target gene promoters, suggesting the existence of an alternative mechanism that compensates for the transcription of genes whose sustained expression is important for critical cellular functions. This work shifts the current paradigm and lays the foundation for studies on the differential roles of PHF20 and PHF20L1 in regulating NSL complex activity in physiological and diseases states.

Histone acetylation of bile acid transporter genes plays a critical role in cirrhosis

Owing to the lack of genetic animal models that adequately recreate key clinical characteristics of cirrhosis, the molecular pathogenesis of cirrhosis has been poorly characterized, and treatments remain limited. Hence, we aimed to better elucidate the pathological mechanisms of cirrhosis using a novel murine model. We report on the first murine genetic model mimicking human cirrhosis induced by hepatocyte-specific elimination of microspherule protein 1 (MCRS1), a member of non-specific lethal (NSL) and INO80 chromatin-modifier complexes. Using this genetic tool with other mouse models, cell culture and human samples, combined with quantitative proteomics, single nuclei/cell RNA sequencing and chromatin immunoprecipitation assays, we investigated mechanisms of cirrhosis. MCRS1 loss in mouse hepatocytes modulates the expression of bile acid (BA) transporters - with a pronounced downregulation of Na+-taurocholate cotransporting polypeptide (NTCP) - concentrating BAs in sinusoids and thereby activating hepatic stellate cells (HSCs) via the farnesoid X receptor (FXR), which is predominantly expressed in human and mouse HSCs. Consistently, re-expression of NTCP in mice reduces cirrhosis, and genetic ablation of FXR in HSCs suppresses fibrotic marks in mice and invitro cell culture. Mechanistically, deletion of a putative SANT domain from MCRS1 evicts histone deacetylase 1 from its histone H3 anchoring sites, increasing histone acetylation of BA transporter genes, modulating their expression and perturbing BA flow. Accordingly, human cirrhosis displays decreased nuclear MCRS1 and NTCP expression. Our data reveal a previously unrecognized function of MCRS1 as a critical histone acetylation regulator, maintaining gene expression and liver homeostasis. MCRS1 loss induces acetylation of BA transporter genes, perturbation of BA flow, and consequently, FXR activation in HSCs. This axis represents a central and universal signaling event in cirrhosis, which has significant implications for cirrhosis treatment. By genetic ablation of MCRS1 in mouse hepatocytes, we generate the first genetic mouse model of cirrhosis that recapitulates human features. Herein, we demonstrate that the activation of the bile acid/FXR axis in liver fibroblasts is key in cirrhosis development.

Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of vasculature.

Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syndromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex-Mof, Kansl2 or Kansl3-unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)-NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work establishes that neurovascular function is determined by the neural metabolic environment.

Evolutionary conserved NSL complex/BRD4 axis controls transcription activation via histone acetylation

Cells rely on a diverse repertoire of genes for maintaining homeostasis, but the transcriptional networks underlying their expression remain poorly understood. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator. It is essential in Drosophila, and haploinsufficiency of the human KANSL1 subunit results in the Koolen-de Vries syndrome. Here, we perform a genome-wide RNAi screen and identify the BET protein BRD4 as an evolutionary conserved co-factor of the NSL complex. Using Drosophila and mouse embryonic stem cells, we characterise a recruitment hierarchy, where NSL-deposited histone acetylation enables BRD4 recruitment for transcription of constitutively active genes. Transcriptome analyses in Koolen-de Vries patient-derived fibroblasts reveals perturbations with a cellular homeostasis signature that are evoked by the NSL complex/BRD4 axis. We propose that BRD4 represents a conserved bridge between the NSL complex and transcription activation, and provide a new perspective in the understanding of their functions in healthy and diseased states.

NSL complex acetylates Lamin A/C.

The non-specific lethal (NSL) complex is a highly conserved MOF acetyltransferase-containing complex. A recent study now shows that the NSL complex acetylates a new non-histone target, Lamin A/C and reveals the NSL complex as a crucial factor in maintaining nuclear architecture and genome integrity.

RNAi-mediated depletion of the NSL complex subunits leads to abnormal chromosome segregation and defective centrosome duplication in Drosophila mitosis.

The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins.

The NSL complex-mediated nucleosome landscape is required to maintain transcription fidelity and suppression of transcription noise.

Nucleosomal organization at gene promoters is critical for transcription, with a nucleosome-depleted region (NDR) at transcription start sites (TSSs) being required for transcription initiation. How NDRs and the precise positioning of the +1 nucleosomes are maintained on active genes remains unclear. Here, we report that the Drosophila nonspecific lethal (NSL) complex is necessary to maintain this stereotypical nucleosomal organization at promoters. Upon NSL1 depletion, nucleosomes invade the NDRs at TSSs of NSL-bound genes. NSL complex member NSL3 binds to TATA-less promoters in a sequence-dependent manner. The NSL complex interacts with the NURF chromatin remodeling complex and is necessary and sufficient to recruit NURF to target promoters. Not only is the NSL complex essential for transcription, but it is required for accurate TSS selection for genes with multiple TSSs. Furthermore, loss of the NSL complex leads to an increase in transcriptional noise. Thus, the NSL complex establishes a canonical nucleosomal organization that enables transcription and determines TSS fidelity.

O-Linked N-acetylglucosamine transferase 1 regulates global histone H4 acetylation via stabilization of the nonspecific lethal protein NSL3

The human males absent on the first (MOF)-containing histone acetyltransferase nonspecific lethal (NSL) complex comprises nine subunits including the O-linked N-acetylglucosamine (O-GlcNAc) transferase, isoform 1 (OGT1). However, whether the O-GlcNAc transferase activity of OGT1 controls histone acetyltransferase activity of the NSL complex and whether OGT1 physically interacts with the other NSL complex subunits remain unclear. Here, we demonstrate that OGT1 regulates the activity of the NSL complex by mainly acetylating histone H4 Lys-16, Lys-5, and Lys-8 via O-GlcNAcylation and stabilization of the NSL complex subunit NSL3. Knocking down or overexpressing OGT1 in human cells remarkably affected the global acetylation of histone H4 residues Lys-16, Lys-5, and Lys-8. Because OGT1 is a subunit of the NSL complex, we also investigated the function of OGT1 in this complex. Co-transfection/co-immunoprecipitation experiments combined with in vitro O-GlcNAc transferase assays confirmed that OGT1 specifically binds to and O-GlcNAcylates NSL3. In addition, wheat germ agglutinin affinity purification verified the occurrence of O-GlcNAc modification on NSL3 in cells. Moreover, O-GlcNAcylation of NSL3 by wild-type OGT1 (OGT1-WT) stabilized NSL3. This stabilization was lost after co-transfection of NSL3 with an OGT1 mutant, OGT1C964A, that lacks O-GlcNAc transferase activity. Furthermore, stabilization of NSL3 by OGT1-WT significantly increased the global acetylation levels of H4 Lys-5, Lys-8, and Lys-16 in cells. These results suggest that OGT1 regulates the activity of the NSL complex by stabilizing NSL3.

NUP98 Fusion Proteins Interact with the NSL and MLL1 Complexes to Drive Leukemogenesis

The nucleoporin 98 gene (NUP98) is fused to a variety of partner genes in multiple hematopoietic malignancies. Here, we demonstrate that NUP98 fusion proteins, including NUP98-HOXA9 (NHA9), NUP98-HOXD13 (NHD13), NUP98-NSD1, NUP98-PHF23, and NUP98-TOP1 physically interact with mixed lineage leukemia 1 (MLL1) and the non-specific lethal (NSL) histone-modifying complexes. Chromatin immunoprecipitation sequencing illustrates that NHA9 and MLL1 co-localize on chromatin and are found associated with Hox gene promoter regions. Furthermore, MLL1 is required for the proliferation of NHA9 cells invitro and invivo. Inactivation of MLL1 leads to decreased expression of genes bound by NHA9 and MLL1 and reverses a gene expression signature found in NUP98-rearranged human leukemias. Our data reveal a molecular dependency on MLL1 function in NUP98-fusion-driven leukemogenesis.

PHF20 Readers Link Methylation of Histone H3K4 and p53 with H4K16 Acetylation

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.

The NSL Chromatin-Modifying Complex Subunit KANSL2 Regulates Cancer Stem-like Properties in Glioblastoma That Contribute to Tumorigenesis.

KANSL2 is an integral subunit of the nonspecific lethal (NSL) chromatin-modifying complex that contributes to epigenetic programs in embryonic stem cells. In this study, we report a role for KANSL2 in regulation of stemness in glioblastoma (GBM), which is characterized by heterogeneous tumor stem-like cells associated with therapy resistance and disease relapse. KANSL2 expression is upregulated in cancer cells, mainly at perivascular regions of tumors. RNAi-mediated silencing of KANSL2 in GBM cells impairs their tumorigenic capacity in mouse xenograft models. In clinical specimens, we found that expression levels of KANSL2 correlate with stemness markers in GBM stem-like cell populations. Mechanistic investigations showed that KANSL2 regulates cell self-renewal, which correlates with effects on expression of the stemness transcription factor POU5F1. RNAi-mediated silencing of POU5F1 reduced KANSL2 levels, linking these two genes to stemness control in GBM cells. Together, our findings indicate that KANSL2 acts to regulate the stem cell population in GBM, defining it as a candidate GBM biomarker for clinical use. Cancer Res; 76(18); 5383-94. ©2016 AACR.

Abstract 883: NUP98-fusion proteins interact with the NSL/MLL1 complexes to drive leukemogenesis

Abstract The Nucleoporin 98 gene (NUP98) is fused to a variety of partner genes in an array of hematopoietic malignancies via chromosomal translocations involving 11p15. NUP98-rearranged leukemias show elevated HOXA and HOXB cluster genes and mouse model systems have recapitulated this high-level expression independent of whether the leukemias are derived from mouse or human bone marrow. However, the molecular mechanisms of NUP98-fusion mediated leukemogenesis and elevated HOX gene expression in this leukemia are unclear. Recent studies in Drosophila show that nucleoplasmic Nup98 functions as a potential transcriptional activator, and physically interacts with the non-specific lethal (NSL) and trithorax (Trx)/mixed lineage leukemia (MLL) complex (Kalverda et al., 2010; Pascual-Garcia et al., 2014). To test whether the intranuclear localized NUP98-fusion proteins interact with NSL/MLL1 complexes, coimmunoprecipitation (co-IP) experiments using 293T cells with overexpression of NUP98 fusions and wild type (WT) NUP98 were performed. NUP98-fusion proteins NUP98-HOXA9, NUP98-HOXD13, NUP98-NSD1 and NUP98-PHF23, but not the full-length WT NUP98, show physical interaction with MLL1 and the NSL histone acetyltransferase (HAT) complexes. Genome-wide chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) illustrated that NUP98-HOXA9 and MLL1 co-localize at Hoxa and Hoxb gene promoters, which correlates with the presence of activating chromatin modifications such as H4K16ac and H3K4me3. In vitro and in vivo functional assays further showed that Mll1 is crucial for the growth of NUP98-HOXA9-transformed cells, and for the initiation and maintenance of NUP98-HOXA9 driven AML. These findings were further supported by transcriptome analyses performed in mouse NUP98-HOXA9 transformed cells lacking Mll1. We found a significant enrichment of co-bound targets of MLL1 and NUP98-HOXA9 and genes downregulated in the absence of Mll1. Gene set enrichment analysis (GSEA) demonstrated strong similarity between Mll1-dependent gene expression signature in our murine NUP98-HOXA9 AML model and the expression profile of human NUP98-fusion AML, indicating that our findings in murine AML models can be extended to human AML. Finally, the overexpression of Hoxa9 and Meis1, direct binding targets of NUP98-HOXA9 and MLL1 that are downregulated upon Mll1 loss, rescues the in vitro transformation defects in NUP98-HOXA9 Mll1-/- cells. In conclusion, our findings support a model where NUP98-fusion proteins recruit the NSL/MLL1 complex as an important step during leukemogenesis. The deregulated HOX gene expression in NUP98 fusion transformed cells remains dependent on MLL1. The discovery of this common leukemogenic pathway for NUP98-fusion proteins opens up new avenues for potential therapeutic opportunities to treat patients with leukemia that harbor various NUP98 rearrangements. Citation Format: Haiming Xu, Daria G. Valerio, Meghan E. Eisold, Amit Sinha, Richard. P. Koche, Wenhuo Hu, Chun-Wei Chen, Scott H. Chu, Gerard L. Brien, Scott A. Armstrong. NUP98-fusion proteins interact with the NSL/MLL1 complexes to drive leukemogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 883.

C-Jun recruits the NSL complex to regulate its target gene expression by modulating H4K16 acetylation and promoting the release of the repressive NuRD complex.

The proto-oncogene c-Jun plays essential roles in various cellular processes, including cell proliferation, cell differentiation, and cellular apoptosis. Enormous efforts have been made to understand the mechanisms regulating c-Jun activation. The males absent on the first (MOF)-containing non-specific lethal (NSL) complex has been shown to positively regulate gene expression. However, the biological function of the NSL complex is largely unknown. Here we present evidence showing that c-Jun recruits the NSL complex to c-Jun target genes upon activation. The NSL complex catalyzes H4K16 acetylation at c-Jun target genes, thereby promoting c-Jun target gene transcription. More interestingly, we also found that the NSL complex promotes the release of the repressive NuRD complex from c-Jun target genes, thus activating c-Jun. Our findings not only reveal a new mechanism regulating c-Jun activation, but also identify the NSL complex as a c-Jun co-activator in c-Jun-regulated gene expression, expanding our knowledge of the function of the NSL complex in gene expression regulation.

Structural analysis of the KANSL1/WDR5/KANSL2 complex reveals that WDR5 is required for efficient assembly and chromatin targeting of the NSL complex.

The subunits of the nonspecific lethal (NSL) complex, which include the histone acetyltransferase MOF (males absent on the first), play important roles in various cellular functions, including transcription regulation and stem cell identity maintenance and reprogramming, and are frequently misregulated in disease. Here, we provide the first biochemical and structural insights into the molecular architecture of this large multiprotein assembly. We identified several direct interactions within the complex and show that KANSL1 acts as a scaffold protein interacting with four other subunits, including WDR5, which in turn binds KANSL2. Structural analysis of the KANSL1/WDR5/KANSL2 subcomplex reveals how WDR5 is recruited into the NSL complex via conserved linear motifs of KANSL1 and KANSL2. Using structure-based KANSL1 mutants in transgenic flies, we show that the KANSL1-WDR5 interaction is required for proper assembly, efficient recruitment of the NSL complex to target promoters, and fly viability. Our data clearly show that the interactions of WDR5 with the MOF-containing NSL complex and MLL/COMPASS histone methyltransferase complexes are mutually exclusive. We propose that rather than being a shared subunit, WDR5 plays an important role in assembling distinct histone-modifying complexes with different epigenetic regulatory roles.

Crosstalk between NSL Histone Acetyltransferase and MLL/SET Complexes: NSL Complex Functions in Promoting Histone H3K4 Di-Methylation Activity by MLL/SET Complexes

hMOF (MYST1), a histone acetyltransferase (HAT), forms at least two distinct multiprotein complexes in human cells. The male specific lethal (MSL) HAT complex plays a key role in dosage compensation in Drosophila and is responsible for histone H4K16ac in vivo. We and others previously described a second hMOF-containing HAT complex, the non-specific lethal (NSL) HAT complex. The NSL complex has a broader substrate specificity, can acetylate H4 on K16, K5, and K8. The WD (tryptophan-aspartate) repeat domain 5 (WDR5) and host cell factor 1 (HCF1) are shared among members of the MLL/SET (mixed-lineage leukemia/set-domain containing) family of histone H3K4 methyltransferase complexes. The presence of these shared subunits raises the possibility that there are functional links between these complexes and the histone modifications they catalyze; however, the degree to which NSL and MLL/SET influence one another's activities remains unclear. Here, we present evidence from biochemical assays and knockdown/overexpression approaches arguing that the NSL HAT promotes histone H3K4me2 by MLL/SET complexes by an acetylation-dependent mechanism. In genomic experiments, we identified a set of genes including ANKRD2, that are affected by knockdown of both NSL and MLL/SET subunits, suggested they are co-regulated by NSL and MLL/SET complexes. In ChIP assays, we observe that depletion of the NSL subunits hMOF or NSL1 resulted in a significant reduction of both H4K16ac and H3K4me2 in the vicinity of the ANKRD2 transcriptional start site proximal region. However, depletion of RbBP5 (a core component of MLL/SET complexes) only reduced H3K4me2 marks, but not H4K16ac in the same region of ANKRD2, consistent with the idea that NSL acts upstream of MLL/SET to regulate H3K4me2 at certain promoters, suggesting coordination between NSL and MLL/SET complexes is involved in transcriptional regulation of certain genes. Taken together, our results suggest a crosstalk between the NSL and MLL/SET complexes in cells.