HMGN5 escorts oncogenic STAT3 signaling by regulating chromatin landscape in tumorigenesis of breast cancer

Epigenetic alterations are widely linked with carcinogenesis, therefore becoming emerging therapeutic targets in the treatment of cancers, including breast cancer. HMGNs are nucleosome binding proteins, which regulate chromatin structures in a cell type- and disease-specific manner. However, the roles of HMGNs in the tumorigenesis of breast cancer are less known. In this study, we report that HMGNs are highly expressed in 3D-cultured breast cancer cells. HMGN5, a member of HMGNs, controls the proliferation, invasion and metastasis of breast cancer cells in vitro and in vivo. Clinically, HMGN5 is an unfavorable prognostic marker in patients. Mechanistically, HMGN5 is governed by active STAT3 transcriptionally and further escorts STAT3 to shape oncogenic chromatin landscape and transcriptional program. Lastly, we provide evidence that interference of HMGN5 by nanoparticle-packaged siRNA is potentially an effective approach in breast cancer treatment. Taken together, our findings reveal a novel feed-forward circuit between HMGN5 and STAT3 in promoting breast cancer tumorigenesis and suggest HMGN5 as a novel epigenetic therapeutic-target in STAT3-hyperactive breast cancer.

NucleoMap: a computational tool for identifying nucleosomes in ultra-high resolution contact maps

Although poorly positioned nucleosomes are ubiquitous in the prokaryote genome, they are difficult to identify with existing nucleosome identification methods. Recently available enhanced high-throughput chromatin conformation capture techniques such as Micro-C, DNase Hi-C, and Hi-CO characterize nucleosome-level chromatin proximity, probing the positions of mono-nucleosomes and the spacing between nucleosome pairs at the same time, enabling profiling of nucleosomes in poorly positioned regions. Here we develop a novel computational approach, NucleoMap, to identify nucleosome positioning from ultra-high resolution chromatin contact maps. By integrating nucleosome binding preferences, read density, and pairing information, NucleoMap precisely locates nucleosomes in both eukaryotic and prokaryotic genomes and outperforms existing nucleosome identification methods in sensitivity and specificity. We rigorously characterize genome-wide association in eukaryotes between the spatial organization of mono-nucleosomes and their corresponding histone modifications, protein binding activities, and higher-order chromatin functions. We also predict two tetra-nucleosome folding structures in human embryonic stem cells using machine learning methods and analysis their distribution at different structural and functional regions. Based on the identified nucleosomes, nucleosome contact maps are constructed, reflecting the inter-nucleosome distances and preserving the original data's contact distance profile.

Structural basis of the regulation of the normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2

Dimethylated histone H3 Lys36 (H3K36me2) regulates gene expression, and aberrant H3K36me2 upregulation, resulting from either the overexpression or point mutation of the dimethyltransferase NSD2, is found in various cancers. Here we report the cryo-electron microscopy structure of NSD2 bound to the nucleosome. Nucleosomal DNA is partially unwrapped, facilitating NSD2 access to H3K36. NSD2 interacts with DNA and H2A along with H3. The NSD2 autoinhibitory loop changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed that two oncogenic mutations, E1099K and T1150A, increase NSD2 catalytic turnover. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild-type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing catalytic turnover. Our analyses guide the development of specific inhibitors of NSD2.

PALI1 facilitates DNA and nucleosome binding by PRC2 and triggers an allosteric activation of catalysis

The polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identities. JARID2 is the only accessory subunit of PRC2 that known to trigger an allosteric activation of methyltransferase. Yet, this mechanism cannot be generalised to all PRC2 variants as, in vertebrates, JARID2 is mutually exclusive with most of the accessory subunits of PRC2. Here we provide functional and structural evidence that the vertebrate-specific PRC2 accessory subunit PALI1 emerged through a convergent evolution to mimic JARID2 at the molecular level. Mechanistically, PRC2 methylates PALI1 K1241, which then binds to the PRC2-regulatory subunit EED to allosterically activate PRC2. PALI1 K1241 is methylated in mouse and human cell lines and is essential for PALI1-induced allosteric activation of PRC2. High-resolution crystal structures revealed that PALI1 mimics the regulatory interactions formed between JARID2 and EED. Independently, PALI1 also facilitates DNA and nucleosome binding by PRC2. In acute myelogenous leukemia cells, overexpression of PALI1 leads to cell differentiation, with the phenotype altered by a separation-of-function PALI1 mutation, defective in allosteric activation and active in DNA binding. Collectively, we show that PALI1 facilitates catalysis and substrate binding by PRC2 and provide evidence that subunit-induced allosteric activation is a general property of holo-PRC2 complexes.

Structures and implications of TBP–nucleosome complexes

The TATA box-binding protein (TBP) is highly conserved throughout eukaryotes and plays a central role in the assembly of the transcription preinitiation complex (PIC) at gene promoters. TBP binds and bends DNA, and directs adjacent binding of the transcription factors TFIIA and TFIIB for PIC assembly. Here, we show that yeast TBP can bind to a nucleosome containing the Widom-601 sequence and that TBP–nucleosome binding is stabilized by TFIIA. We determine three cryo-electron microscopy (cryo-EM) structures of TBP–nucleosome complexes, two of them containing also TFIIA. TBP can bind to superhelical location (SHL) –6, which contains a TATA-like sequence, but also to SHL +2, which is GC-rich. Whereas binding to SHL –6 can occur in the absence of TFIIA, binding to SHL +2 is only observed in the presence of TFIIA and goes along with detachment of upstream terminal DNA from the histone octamer. TBP–nucleosome complexes are sterically incompatible with PIC assembly, explaining why a promoter nucleosome generally impairs transcription and must be moved before initiation can occur.

Chromatin landscape signals differentially dictate the activities of mSWI/SNF family complexes

Mammalian SWI/SNF (mSWI/SNF) adenosine triphosphate–dependent chromatin remodelers modulate genomic architecture and gene expression and are frequently mutated in disease. However, the specific chromatin features that govern their nucleosome binding and remodeling activities remain unknown. We subjected endogenously purified mSWI/SNF complexes and their constituent assembly modules to a diverse library of DNA-barcoded mononucleosomes, performing more than 25,000 binding and remodeling measurements. Here, we define histone modification-, variant-, and mutation-specific effects, alone and in combination, on mSWI/SNF activities and chromatin interactions. Further, we identify the combinatorial contributions of complex module components, reader domains, and nucleosome engagement properties to the localization of complexes to selectively permissive chromatin states. These findings uncover principles that shape the genomic binding and activity of a major chromatin remodeler complex family.

ΔNp63 is a pioneer factor that binds inaccessible chromatin and elicits chromatin remodeling

Background ΔNp63 is a master transcriptional regulator playing critical roles in epidermal development and other cellular processes. Recent studies suggest that ΔNp63 functions as a pioneer factor that can target its binding sites within inaccessible chromatin and induce chromatin remodeling. Methods In order to examine if ΔNp63 can bind to inaccessible chromatin and to determine if specific histone modifications are required for binding, we induced ΔNp63 expression in two p63-naïve cell lines. ΔNp63 binding was then examined by ChIP-seq and the chromatin at ΔNp63 targets sites was examined before and after binding. Further analysis with competitive nucleosome binding assays was used to determine how ΔNp63 directly interacts with nucleosomes. Results Our results show that before ΔNp63 binding, targeted sites lack histone modifications, indicating ΔNp63’s capability to bind at unmodified chromatin. Moreover, the majority of the sites that are bound by ectopic ΔNp63 expression exist in an inaccessible state. Once bound, ΔNp63 induces acetylation of the histone and the repositioning of nucleosomes at its binding sites. Further analysis with competitive nucleosome binding assays reveal that ΔNp63 can bind directly to nucleosome edges with significant binding inhibition occurring within 50 bp of the nucleosome dyad. Conclusion Overall, our results demonstrate that ΔNp63 is a pioneer factor that binds nucleosome edges at inaccessible and unmodified chromatin sites and induces histone acetylation and nucleosome repositioning.