ANP32e Binds Histone H2A.Z in a Cell Cycle-Dependent Manner and Regulates Its Protein Stability in the Cytoplasm.

ANP32e, a chaperone of H2A.Z, is receiving increasing attention because of its association with cancer growth and progression. An unanswered question is whether ANP32e regulates H2A.Z dynamics during the cell cycle; this could have clear implications for the proliferation of cancer cells. We confirmed that ANP32e regulates the growth of human U2OS cancer cells and preferentially interacts with H2A.Z during the G1 phase of the cell cycle. Unexpectedly, ANP32e does not mediate the removal of H2A.Z from chromatin, is not a stable component of the p400 remodeling complex and is not strongly associated with chromatin. Instead, most ANP32e is in the cytoplasm. Here, ANP32e preferentially interacts with H2A.Z in the G1 phase in response to an increase in H2A.Z protein abundance and regulates its protein stability. This G1-specific interaction was also observed in the nucleoplasm but was unrelated to any change in H2A.Z abundance. These results challenge the idea that ANP32e regulates the abundance of H2A.Z in chromatin as part of a chromatin remodeling complex. We propose that ANP32e is a molecular chaperone that maintains the soluble pool of H2A.Z by regulating its protein stability and acting as a buffer in response to cell cycle-dependent changes in H2A.Z abundance.

Multi-level 3D genome organization deteriorates during breast cancer progression.

Breast cancer entails intricate alterations in genome organization and expression. However, how three-dimensional (3D) chromatin structure changes in the progression from a normal to a breast cancer malignant state remains unknown. To address this, we conducted an analysis combining Hi-C data with lamina-associated domains (LADs), epigenomic marks, and gene expression in an in vitro model of breast cancer progression. Our results reveal that while the fundamental properties of topologically associating domains (TADs) remain largely stable, significant changes occur in the organization of compartments and subcompartments. These changes are closely correlated with alterations in the expression of oncogenic genes. We also observe a restructuring of TAD-TAD interactions, coinciding with a loss of spatial compartmentalization and radial positioning of the 3D genome. Notably, we identify a previously unrecognized interchromosomal insertion event, wherein a locus on chromosome 8 housing the MYC oncogene is inserted into a highly active subcompartment on chromosome 10. This insertion leads to the formation of de novo enhancer contacts and activation of the oncogene, illustrating how structural variants can interact with the 3D genome to drive oncogenic states. In summary, our findings provide evidence for the degradation of genome organization at multiple scales during breast cancer progression revealing novel relationships between genome 3D structure and oncogenic processes.

The Role of the Histone Variant H2A.Z in Metazoan Development.

During the emergence and radiation of complex multicellular eukaryotes from unicellular ancestors, transcriptional systems evolved by becoming more complex to provide the basis for this morphological diversity. The way eukaryotic genomes are packaged into a highly complex structure, known as chromatin, underpins this evolution of transcriptional regulation. Chromatin structure is controlled by a variety of different epigenetic mechanisms, including the major mechanism for altering the biochemical makeup of the nucleosome by replacing core histones with their variant forms. The histone H2A variant H2A.Z is particularly important in early metazoan development because, without it, embryos cease to develop and die. However, H2A.Z is also required for many differentiation steps beyond the stage that H2A.Z-knockout embryos die. H2A.Z can facilitate the activation and repression of genes that are important for pluripotency and differentiation, and acts through a variety of different molecular mechanisms that depend upon its modification status, its interaction with histone and nonhistone partners, and where it is deposited within the genome. In this review, we discuss the current knowledge about the different mechanisms by which H2A.Z regulates chromatin function at various developmental stages and the chromatin remodeling complexes that determine when and where H2A.Z is deposited.

Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications.

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- or C-termini with segmental isotope labelling for NMR spectroscopy. With access to these precisely-defined variants, we show that PTMs in both the N- and C-termini cause changes in the chemical shifts and conformational populations in regions distant from the PTM sites; up to 50-60 residues upstream of the PTM site. The PTMs investigated had only minor effects on binding of HMGN1 to nucleosome core particles, suggesting that they have other regulatory roles. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the role of PTMs with atomic precision, from both structural and functional perspectives.

Conservation and expansion of a necrosis-inducing small secreted protein family from host-variable phytopathogens of the Sclerotiniaceae.

Fungal effector proteins facilitate host-plant colonization and have generally been characterized as small secreted proteins (SSPs). We classified and functionally tested SSPs from the secretomes of three closely related necrotrophic phytopathogens: Ciborinia camelliae, Botrytis cinerea, and Sclerotinia sclerotiorum. Alignment of predicted SSPs identified a large protein family that share greater than 41% amino acid identity and that have key characteristics of previously described microbe-associated molecular patterns (MAMPs). Strikingly, 73 of the 75 SSP family members were predicted within the secretome of the host-specialist C. camelliae with single-copy homologs identified in the secretomes of the host generalists S. sclerotiorum and B. cinerea. To explore the potential function of this family of SSPs, 10 of the 73 C. camelliae proteins, together with the single-copy homologs from S. sclerotiorum (SsSSP3) and B. cinerea (BcSSP2), were cloned and expressed as recombinant proteins. Infiltration of SsSSP3 and BcSSP2 into host tissue induced rapid necrosis. In contrast, only one of the 10 tested C. camelliae SSPs was able to induce a limited amount of necrosis. Analysis of chimeric proteins consisting of domains from both a necrosis-inducing and a non-necrosis-inducing SSP demonstrated that the C-terminus of the S. sclerotiorum SSP is essential for necrosis-inducing function. Deletion of the BcSSP2 homolog from B. cinerea did not affect growth or pathogenesis. Thus, this research uncovered a family of highly conserved SSPs present in diverse ascomycetes that exhibit contrasting necrosis-inducing functions.