KAP1 Is a Chromatin Reader that Couples Steps of RNA Polymerase II Transcription to Sustain Oncogenic Programs.

Precise control of the RNA polymerase II (RNA Pol II) cycle, including pausing and pause release, maintains transcriptional homeostasis and organismal functions. Despite previous work to understand individual transcription steps, we reveal a mechanism that integrates RNA Pol II cycle transitions. Surprisingly, KAP1/TRIM28 uses a previously uncharacterized chromatin reader cassette to bind hypo-acetylated histone 4 tails at promoters, guaranteeing continuous progression of RNA Pol II entry to and exit from the pause state. Upon chromatin docking, KAP1 first associates with RNA Pol II and then recruits a pathway-specific transcription factor (SMAD2) in response to cognate ligands, enabling gene-selective CDK9-dependent pause release. This coupling mechanism is exploited by tumor cells to aberrantly sustain transcriptional programs commonly dysregulated in cancer patients. The discovery of a factor integrating transcription steps expands the functional repertoire by which chromatin readers operate and provides mechanistic understanding of transcription regulation, offering alternative therapeutic opportunities to target transcriptional dysregulation.

Sonrotoclax overcomes BCL2 G101V mutation-induced venetoclax resistance in preclinical models of hematologic malignancy.

ABSTRACT: Venetoclax, the first-generation inhibitor of the apoptosis regulator B-cell lymphoma 2 (BCL2), disrupts the interaction between BCL2 and proapoptotic proteins, promoting the apoptosis in malignant cells. Venetoclax is the mainstay of therapy for relapsed chronic lymphocytic leukemia and is under investigation in multiple clinical trials for the treatment of various cancers. Although venetoclax treatment can result in high rates of durable remission, relapse has been widely observed, indicating the emergence of drug resistance. The G101V mutation in BCL2 is frequently observed in patients who relapsed treated with venetoclax and sufficient to confer resistance to venetoclax by interfering with compound binding. Therefore, the development of next-generation BCL2 inhibitors to overcome drug resistance is urgently needed. In this study, we discovered that sonrotoclax, a potent and selective BCL2 inhibitor, demonstrates stronger cytotoxic activity in various hematologic cancer cells and more profound tumor growth inhibition in multiple hematologic tumor models than venetoclax. Notably, sonrotoclax effectively inhibits venetoclax-resistant BCL2 variants, such as G101V. The crystal structures of wild-type BCL2/BCL2 G101V in complex with sonrotoclax revealed that sonrotoclax adopts a novel binding mode within the P2 pocket of BCL2 and could explain why sonrotoclax maintains stronger potency than venetoclax against the G101V mutant. In summary, sonrotoclax emerges as a potential second-generation BCL2 inhibitor for the treatment of hematologic malignancies with the potential to overcome BCL2 mutation-induced venetoclax resistance. Sonrotoclax is currently under investigation in multiple clinical trials.

Genetic Control of Alternative Splicing and its Distinct Role in Colorectal Cancer Mechanisms.

BACKGROUND & AIMS: Dysregulation of alternative splicing is implicated in many human diseases, and understanding the genetic variation underlying transcript splicing is essential to dissect the molecular mechanisms of cancers. We aimed to provide a comprehensive functional dissection of splicing quantitative trait loci (sQTLs) in cancer and focus on elucidating its distinct role in colorectal cancer (CRC) mechanisms. METHODS: We performed a comprehensive sQTL analysis to identify genetic variants that control messenger RNA splicing across 33 cancer types from The Cancer Genome Atlas and independently validated in our 154 CRC tissues. Then, large-scale, multicenter, multi-ethnic case-control studies (34,585 cases and 76,023 controls) were conducted to examine the association of these sQTLs with CRC risk. A series of biological experiments in vitro and in vivo were performed to investigate the potential mechanisms of the candidate sQTLs and target genes. RESULTS: The molecular characterization of sQTL revealed its distinct role in cancer susceptibility. Tumor-specific sQTL further showed better response to cancer development. In addition, functionally informed polygenic risk score highlighted the potentiality of sQTLs in the CRC prediction. Complemented by large-scale population studies, we identified that the risk allele (T) of a multi-ancestry-associated sQTL rs61746794 significantly increased the risk of CRC in Chinese (odds ratio, 1.20; 95% CI, 1.12-1.29; P = 8.82 × 10-7) and European (odds ratio, 1.11; 95% CI, 1.07-1.16; P = 1.13 × 10-7) populations. rs61746794-T facilitated PRMT7 exon 16 splicing mediated by the RNA-binding protein PRPF8, thus increasing the level of canonical PRMT7 isoform (PRMT7-V2). Overexpression of PRMT7-V2 significantly enhanced the growth of CRC cells and xenograft tumors compared with PRMT7-V1. Mechanistically, PRMT7-V2 functions as an epigenetic writer that catalyzes the arginine methylation of H4R3 and H3R2, subsequently regulating diverse biological processes, including YAP, AKT, and KRAS pathway. A selective PRMT7 inhibitor, SGC3027, exhibited antitumor effects on human CRC cells. CONCLUSIONS: Our study provides an informative sQTLs resource and insights into the regulatory mechanisms linking splicing variants to cancer risk and serving as biomarkers and therapeutic targets.

Dietary polyphenols link extracellular histones and nonhistone proteins.

Numerous studies have demonstrated antioxidant, anti-inflammatory, antimicrobial, anticancer, and cardio-protective activities of dietary polyphenols, but due to diverse structures and subclasses of polyphenols, little is known about their mechanisms of action. The study by Yamaguchi et al. published in JBC provides mechanistic insights into how dietary polyphenols confer histone-binding ability on certain proteins and motivates the research community to further explore health benefits of polyphenols.

HSPA12A was identified as a key driver in colorectal cancer GWAS loci 10q26.12 and modulated by an enhancer-promoter interaction.

Although genome-wide association studies (GWASs) have identified over 100 colorectal cancer (CRC) risk loci, an understanding of causal genes or risk variants and their biological functions in these loci remain unclear. Recently, genomic loci 10q26.12 with lead SNP rs1665650 was identified as an essential CRC risk loci of Asian populations. However, the functional mechanism of this region has not been fully clarified. Here, we applied an RNA interfering-based on-chip approach to screen for the genes essential for cell proliferation in the CRC risk loci 10q26.12. Notably, HSPA12A had the most significant effect among the identified genes and functioned as a crucial oncogene facilitating cell proliferation. Moreover, we conducted an integrative fine-mapping analysis to identify putative casual variants and further explored their association with CRC risk in a large-scale Chinese population consisting of 4054 cases and 4054 controls and also independently validated in 5208 cases and 20,832 controls from the UK biobank cohort. We identified a risk SNP rs7093835 in the intron of HSPA12A that was significantly associated with an increased risk of CRC (OR 1.23, 95% CI 1.08-1.41, P = 1.92 × 10-3). Mechanistically, the risk variant could facilitate an enhancer-promoter interaction mediated by the transcriptional factor (TF) GRHL1 and ultimately upregulate HSPA12A expression, which provides functional evidence to support our population findings. Collectively, our study reveals the important role of HSPA12A in CRC development and illustrates a novel enhancer-promoter interaction module between HSPA12A and its regulatory elements rs7093835, providing new insights into the etiology of CRC.

Inhibitors against Two PDZ Domains of MDA-9 Suppressed Migration of Breast Cancer Cells.

Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein-ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future.

New insights into M1/M2 macrophages: key modulators in cancer progression.

Infiltration of macrophages in and around tumor nest represents one of the most crucial hallmarks during tumor progression. The mutual interactions with tumor cells and stromal microenvironment contribute to phenotypically polarization of tumor associated macrophages. Macrophages consist of at least two subgroups, M1 and M2. M1 phenotype macrophages are tumor-resistant due to intrinsic phagocytosis and enhanced antitumor inflammatory reactions. Contrastingly, M2 are endowed with a repertoire of tumor-promoting capabilities involving immuno-suppression, angiogenesis and neovascularization, as well as stromal activation and remodeling. The functional signature of M2 incorporates location-related, mutually connected, and cascade-like reactions, thereby accelerating paces of tumor aggressiveness and metastasis. In this review, mechanisms underlying the distinct functional characterization of M1 and M2 macrophages are demonstrated to make sense of M1 and M2 as key regulators during cancer progression.

Subtotal parathyroidectomy versus total parathyroidectomy with autotransplantation for secondary hyperparathyroidism: an updated systematic review and meta-analysis.

PURPOSE: The optimal surgical approach of parathyroidectomy for patients with secondary hyperparathyroidism (SHPT) has been controversial. The updated meta-analysis aimed to compare the effectiveness of subtotal parathyroidectomy (SPTX) versus total parathyroidectomy with autotransplantation (TPTX + AT). METHODS: A thorough systematic search was performed on the databases of PubMed, EMBASE, and Cochrane library to identify eligible studies. Data were extracted and pooled into a meta-analysis. The primary outcomes were the symptomatic improvement, radiological changes, hypocalcemia rate, the requirement for vitamin D analogues, time to recurrence, recurrence, persistence, and reoperation rates of SPTX versus TPTX + AT. RESULTS: A total of 18 studies with 3656 patients (1864 patients in SPTX and 1792 patients in TPTX + AT group) were included, and 15 studies were included in quantitative synthesis. No significant difference was observed in symptomatic improvement (93.3%, 89.0%; P = 0.99), radiological changes (85.4%, 85.3%; P = 0.91), hypocalcemia rate (16.6%, 18.1%; P = 0.29), persistence rate (6.1%, 2.0%; P = 0.16), time to recurrence (mean difference 1.46; P = 0.87), recurrence rate (9.2%, 7.1%; P = 0.76), and reoperation rate (5.3%, 5.8%; P = 0.66) between SPTX and TPTX + AT groups. Longer operative time (150 vs. 120 min), prolonged in-hospital stay (5.0 vs. 4.1 days), lower 1-month serum calcium level, and higher requirement for vitamin D analogues at 12 months were significantly observed in patients who underwent TPTX + AT compared to SPTX. CONCLUSIONS: The two surgical approaches were both effective at controlling SHPT in clinical and laboratory terms. However, most of the data shown were not statistically significant. It was acceptable that surgeons chose either SPTX or TPTX + AT for SHPT.

Structural basis for the specific recognition of 18S rRNA by APUM23.

PUF (Pumilio/fem-3 mRNA binding factor) proteins, a conserved family of RNA-binding proteins, recognize specific single-strand RNA targets in a specific modular way. Although plants have a greater number of PUF protein members than do animal and fungal systems, they have been the subject of fewer structural and functional investigations. The aim of this study was to elucidate the involvement of APUM23, a nucleolar PUF protein in the plant Arabidopsis, in pre-rRNA processing. APUM23 is distinct from classical PUF family proteins, which are located in the cytoplasm and bind to 3'UTRs of mRNA to modulate mRNA expression and localization. We found that the complete RNA target sequence of APUM23 comprises 11 nt in 18S rRNA at positions 1141-1151. The complex structure shows that APUM23 has 10 PUF repeats; it assembles into a C-shape, with an insertion located within the inner concave surface. We found several different RNA recognition features. A notable structural feature of APUM23 is an insertion in the third PUF repeat that participates in nucleotide recognition and maintains the correct conformation of the target RNA. Our findings elucidate the mechanism for APUM23's-specific recognition of 18S rRNA.

Stromal fibroblast activation protein alpha promotes gastric cancer progression via epithelial-mesenchymal transition through Wnt/ ß-catenin pathway.

BACKGROUND: To investigate the influence of fibroblast activation protein alpha (FAP) derived from cancer-associated fibroblasts (CAFs), as well as potential mechanism of epithelial mesenchymal transition (EMT), on gastric cancer (GC) progression. METHODS: Correlation between CAFs-derived FAP and clinical results has been studied by using 60 GC cases. To confirm this relationship, SGC7901 cells were co-cultured with pre-established FAP-overexpressed fibroblasts in vitro and the characteristics including proliferation, migration, invasion and apoptosis abilities were detected subsequently. Meanwhile, SGC and GES1 cells cocultured with FAP-overexpressed fibroblasts were treated with cis-platinum for apoptotic analysis. The underlying EMT was detected by analyzing expression level of E-cadherin, ZO-1, N-cadherin, Vimentin, α-SMA, DKK1 and LEF-1 through western blot and immunofluorescence staining assay. Finally, the tumor-promoting ability of FAP was investigated by utlizing a xenograft gastric cancer nude mouse model. RESULTS: It show that FAP has a high-risk correlation with the malignant level of clinical outcomes in GC patients. FAP promotes the ability of proliferation, migration, invasion, apoptosis-inhibition of SGC7901 cells and induces apoptosis of GES1 cells in vitro. The mechanism study shows that epithelial markers have been down-regulated and mesenchymal markers and Wnt/ß-catenin signal pathway related proteins have been up-regulated. Animal assay suggests that tumor burden has been enhanced by FAP significantly in vivo. CONCLUSIONS: Stromal FAP could be a potential prognostic biomarker in GC by promoting cancer progression via EMT through Wnt/ ß-catenin signal pathway.

NMR characterization of weak interactions between RhoGDI2 and fragment screening hits.

BACKGROUND: The delineation of intrinsically weak interactions between novel targets and fragment screening hits has long limited the pace of hit-to-lead evolution. Rho guanine-nucleotide dissociation inhibitor 2 (RhoGDI2) is a novel target that lacks any chemical probes for the treatment of tumor metastasis. METHODS: Protein-observed and ligand-observed NMR spectroscopy was used to characterize the weak interactions between RhoGDI2 and fragment screening hits. RESULTS: We identified three hits of RhoGDI2 using streamlined NMR fragment-based screening. The binding site residues were assigned using non-uniformly sampled Cα- and Hα-based three dimensional NMR spectra. The molecular docking to the proposed geranylgeranyl binding pocket of RhoGDI2 was guided by NMR restraints of chemical shift perturbations and ligand-observed transferred paramagnetic relaxation enhancement. We further validated the weak RhoGDI2-hit interactions using mutagenesis and structure-affinity analysis. CONCLUSIONS: Weak interactions between RhoGDI2 and fragment screening hits were delineated using an integrated NMR approach. GENERAL INTERESTS: Binders to RhoGDI2 as a potential anti-cancer target have been first reported, and their weak interactions were depicted using NMR spectroscopy. Our work highlights the powerfulness and the versatility of the integrative NMR techniques to provide valuable structural insight into the intrinsically weak interactions between RhoGDI2 and the fragment screening hits, which could hardly be conceived using other biochemical techniques.

Fluorine Pseudocontact Shifts Used for Characterizing the Protein-Ligand Interaction Mode in the Limit of NMR Intermediate Exchange.

The characterization of protein-ligand interaction modes becomes recalcitrant in the NMR intermediate exchange regime as the interface resonances are broadened beyond detection. Here, we determined the 19 F low-populated bound-state pseudocontact shifts (PCSs) of mono- and di-fluorinated inhibitors of the BRM bromodomain using a highly skewed protein/ligand ratio. The bound-state 19 F PCSs were retrieved from 19 F chemical exchange saturation transfer (CEST) in the presence of the lanthanide-labeled protein, which was termed the 19 F PCS-CEST approach. These PCSs enriched in spatial information enabled the identification of best-fitting poses, which agree well with the crystal structure of a more soluble analog in complex with the BRM bromodomain. This approach fills the gap of the NMR structural characterization of lead-like inhibitors with moderate affinities to target proteins, which are essential for structure-guided hit-to-lead evolution.

Milky spots: omental functional units and hotbeds for peritoneal cancer metastasis.

As the most common metastatic disease of abdomen pelvic cavity cancer, peritoneal carcinomatosis (PC) renders significant negative impact on patient survival and quality of life. Invasive peritoneal exfoliated cancer cells (PECCs) preferentially select the omentum as a predominant target site for cancer cell colonization and proliferation compared with other tissues in the abdominal cavity. The precise pathogenic mechanism remains to be determined. As omental milky spots (MSs) are the major implantation site for malignant cells in peritoneal dissemination, researches on mechanisms of PC have been mainly focused on MS, primitive lymphoid tissues with unique structural features, and functional characteristics. To date, extensive biophysical and biochemical methods have been manipulated to investigate the MS exact function in the peritoneal cavity. This review summarized MS as hotbeds for PECC. The anatomical distribution was briefly described first. Then, MS histology was systematically reviewed, including morphological features, cellular constituents, and histological staining methods. At last, the roles of MS in PC pathological process were summarized with special emphasis on the distinct roles of macrophages.

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.

Aptamer-based sample purification for mass spectrometric quantification of trastuzumab in human serum.

In this study, we developed a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify trastuzumab in human serum using aptamers for sample purification. Trastuzumab was extracted from serum samples using the capture probe based on its aptamer CH1S-3, followed by reduction, alkylation, trypsin digestion, and quantification using LC-MS/MS. Additionally, a unique peptide, FTISADTSK, was employed as a surrogate peptide and quantified, and *FTISADTSK (13C915N-labeled phenylalanine) was used as an internal standard to minimize variability in detection among the samples. The detection range for this method was 0.5-250 µg/mL, with a high correlation coefficient (r2 > 0.99). The intra- and inter-day precision (%CV, the coefficient of variation) of the quality control samples was less than 12.7%, and the accuracy (%bias) was below 8.64%. After optimization and verification, this assay was used to determine trastuzumab levels in clinical human serum samples. The results indicated that the trastuzumab concentrations had an approximate 4-fold difference among ten patients (range: 11.80-41.90 µg/mL). This study provides a novel approach for the accurate and quantitative monitoring of the mAb-trastuzumab.

Molecular insight into the PCNA-binding mode of FBH1.

F-box DNA helicase 1 (FBH1) is involved in the regulation of cell responses to replicative stress. FBH1 is recruited to stalled DNA replication fork by PCNA where it inhibits homologous recombination and catalyzes fork regression. Here, we report the structural basis for the molecular recognition of two distinctly different motifs of FBH1, FBH1PIP and FBH1APIM, by PCNA. The crystal structure of PCNA in complex with FBH1PIP and analysis of NMR perturbations reveal overlapped FBH1PIP and FBH1APIM binding sites of PCNA and the dominant contribution of FBH1PIP in this interaction.

Genetic Variants That Impact Alternative Polyadenylation in Cancer Represent Candidate Causal Risk Loci.

Alternative polyadenylation (APA) is emerging as a major mechanism of posttranscriptional regulation. APA can impact the development and progression of cancer, suggesting that the genetic determinants of APA might play an important role in regulating cancer risk. Here, we depicted a pan-cancer atlas of human APA quantitative trait loci (apaQTL), containing approximately 0.7 million apaQTLs across 32 cancer types. Systematic multiomics analyses indicated that cancer apaQTLs could contribute to APA regulation by altering poly(A) motifs, RNA-binding proteins (RBP), and chromatin regulatory elements and were preferentially enriched in genome-wide association studies (GWAS)-identified cancer susceptibility loci. Moreover, apaQTL-related genes (aGene) were broadly related to cancer signaling pathways, high mutational burden, immune infiltration, and drug response, implicating their potential as therapeutic targets. Furthermore, apaQTLs were mapped in Chinese colorectal cancer tumor tissues and then screened for functional apaQTLs associated with colorectal cancer risk in 17,789 cases and 19,951 controls using GWAS-ChIP data, with independent validation in a large-scale population consisting of 6,024 cases and 10,022 controls. A multi-ancestry-associated apaQTL variant rs1020670 with a C>G change in DNM1L was identified, and the G allele contributed to an increased risk of colorectal cancer. Mechanistically, the risk variant promoted aberrant APA and facilitated higher usage of DNM1L proximal poly(A) sites mediated by the RBP CSTF2T, which led to higher expression of DNM1L with a short 3'UTR. This stabilized DNM1L to upregulate its expression, provoking colorectal cancer cell proliferation. Collectively, these findings generate a resource for understanding APA regulation and the genetic basis of human cancers, providing insights into cancer etiology. SIGNIFICANCE: Cancer risk is mediated by alternative polyadenylation quantitative trait loci, including the rs1020670-G variant that promotes alternative polyadenylation of DNM1L and increases colorectal cancer risk.

Genome-wide enhancer-gene regulatory maps link causal variants to target genes underlying human cancer risk.

Genome-wide association studies have identified numerous variants associated with human complex traits, most of which reside in the non-coding regions, but biological mechanisms remain unclear. However, assigning function to the non-coding elements is still challenging. Here we apply Activity-by-Contact (ABC) model to evaluate enhancer-gene regulation effect by integrating multi-omics data and identified 544,849 connections across 20 cancer types. ABC model outperforms previous approaches in linking regulatory variants to target genes. Furthermore, we identify over 30,000 enhancer-gene connections in colorectal cancer (CRC) tissues. By integrating large-scale population cohorts (23,813 cases and 29,973 controls) and multipronged functional assays, we demonstrate an ABC regulatory variant rs4810856 associated with CRC risk (Odds Ratio = 1.11, 95%CI = 1.05-1.16, P = 4.02 × 10-5) by acting as an allele-specific enhancer to distally facilitate PREX1, CSE1L and STAU1 expression, which synergistically activate p-AKT signaling. Our study provides comprehensive regulation maps and illuminates a single variant regulating multiple genes, providing insights into cancer etiology.

The origins of resident macrophages in mammary gland influence the tumorigenesis of breast cancer.

Macrophage is the important sentinel cell type of innate immune system, and bridge with the adaptive immune response via antigen presentation. Tissue-resident macrophages are universal in almost all organs and play essential roles in maintaining specific organ homeostasis, inflammation responses, and disease genesis, including tumorigenesis. Macrophage is generally divided into two extreme statuses, M1 and M2, with sophisticated continuous subtypes due to different stimuli and microenvironments. Tumor-associated macrophage (TAM) is regarded as the key factor related to the prognosis, staging, classification, and treatment strategy of various cancers. However, emerging evidence indicated potential opposite functions of TAM in different tumor models. Recent studies found that different originated resident macrophages show notably different profiles in the same tissue niche. More evidence pointed out that the strategies to repolarize the subtypes of TAM or resident macrophages are valuable in carcinoma treatments. In the breast cancer model, studies pointed that macrophages located differently in histology show obvious different cell markers and functions. In this review, we will illustrate the profiles of resident macrophages in breast cancer with various aspects, including origination, polarization, tumoricidal activity, tumorigenesis, and the factors that could regulate the functions of macrophages.

Structural basis for binding diversity of acetyltransferase p300 to the nucleosome.

p300 is a human acetyltransferase that associates with chromatin and mediates vital cellular processes. We now report the cryo-electron microscopy structures of the p300 catalytic core in complex with the nucleosome core particle (NCP). In the most resolved structure, the HAT domain and bromodomain of p300 contact nucleosomal DNA at superhelical locations 2 and 3, and the catalytic site of the HAT domain are positioned near the N-terminal tail of histone H4. Mutations of the p300-DNA interfacial residues of p300 substantially decrease binding to NCP. Three additional classes of p300-NCP complexes show different modes of the p300-NCP complex formation. Our data provide structural details critical to our understanding of the mechanism by which p300 acetylates multiple sites on the nucleosome.