The high mobility group protein HMG20A cooperates with the histone reader PHF14 to modulate TGFß and Hippo pathways.

High mobility group (HMG) proteins are chromatin regulators with essential functions in development, cell differentiation and cell proliferation. The protein HMG20A is predicted by the AlphaFold2 software to contain three distinct structural elements, which we have functionally characterized: i) an amino-terminal, intrinsically disordered domain with transactivation activity; ii) an HMG box with higher binding affinity for double-stranded, four-way-junction DNA than for linear DNA; and iii) a long coiled-coil domain. Our proteomic study followed by a deletion analysis and structural modeling demonstrates that HMG20A forms a complex with the histone reader PHF14, via the establishment of a two-stranded alpha-helical coiled-coil structure. siRNA-mediated knockdown of either PHF14 or HMG20A in MDA-MB-231 cells causes similar defects in cell migration, invasion and homotypic cell-cell adhesion ability, but neither affects proliferation. Transcriptomic analyses demonstrate that PHF14 and HMG20A share a large subset of targets. We show that the PHF14-HMG20A complex modulates the Hippo pathway through a direct interaction with the TEAD1 transcription factor. PHF14 or HMG20A deficiency increases epithelial markers, including E-cadherin and the epithelial master regulator TP63 and impaired normal TGFß-trigged epithelial-to-mesenchymal transition. Taken together, these data indicate that PHF14 and HMG20A cooperate in regulating several pathways involved in epithelial-mesenchymal plasticity.

The Histone Variant MacroH2A1 Regulates Key Genes for Myogenic Cell Fusion in a Splice-Isoform Dependent Manner.

MacroH2A histone variants have functions in differentiation, somatic cell reprogramming and cancer. However, at present, it is not clear how macroH2As affect gene regulation to exert these functions. We have parted from the initial observation that loss of total macroH2A1 led to a change in the morphology of murine myotubes differentiated ex vivo. The fusion of myoblasts to myotubes is a key process in embryonic myogenesis and highly relevant for muscle regeneration after acute or chronic injury. We have focused on this physiological process, to investigate the functions of the two splice isoforms of macroH2A1. Individual perturbation of the two isoforms in myotubes forming in vitro from myogenic C2C12 cells showed an opposing phenotype, with macroH2A1.1 enhancing, and macroH2A1.2 reducing, fusion. Differential regulation of a subset of fusion-related genes encoding components of the extracellular matrix and cell surface receptors for adhesion correlated with these phenotypes. We describe, for the first time, splice isoform-specific phenotypes for the histone variant macroH2A1 in a physiologic process and provide evidence for a novel underlying molecular mechanism of gene regulation.

Hydrolysis of ADP-Ribosylation by Macrodomains.

ADP-ribosylation is the process of transferring the ADP-ribose moiety from NAD+ to a substrate. While a number of proteins represent well described substrates accepting ADP-ribose modification, a recent report demonstrated biological role for DNA ADP-ribosylation as well. The conserved macrodomain fold of several known hydrolyses was found to possess de-ADP-ribosylating activity and the ability to hydrolyze (reverse) ADP-ribosylation. Here we summarize the methods that can be employed to study mono-ADP-ribosylation hydrolysis by macrodomains.

MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption.

Histone variants are structural components of eukaryotic chromatin that can replace replication-coupled histones in the nucleosome. The histone variant macroH2A1.1 contains a macrodomain capable of binding NAD+-derived metabolites. Here we report that macroH2A1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing, and that myotubes that lack macroH2A1.1 have a defect in mitochondrial respiratory capacity. We found that the metabolite-binding macrodomain was essential for sustained optimal mitochondrial function but dispensable for gene regulation. Through direct binding, macroH2A1.1 inhibits basal poly-ADP ribose polymerase 1 (PARP-1) activity and thus reduces nuclear NAD+ consumption. The resultant accumulation of the NAD+ precursor NMN allows for maintenance of mitochondrial NAD+ pools that are critical for respiration. Our data indicate that macroH2A1.1-containing chromatin regulates mitochondrial respiration by limiting nuclear NAD+ consumption and establishing a buffer of NAD+ precursors in differentiated cells.

MacroH2A histone variants maintain nuclear organization and heterochromatin architecture.

Genetic loss-of-function studies on development, cancer and somatic cell reprogramming have suggested that the group of macroH2A histone variants might function through stabilizing the differentiated state by a yet unknown mechanism. Here, we present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences. We further identify macroH2A on sites of interstitial heterochromatin decorated by histone H3 trimethylated on K9 (H3K9me3). Loss of macroH2A leads to major defects in nuclear organization, including reduced nuclear circularity, disruption of nucleoli and a global loss of dense heterochromatin. Domains formed by DNA repeat sequences are disorganized, expanded and fragmented, and mildly re-expressed when depleted of macroH2A. At the molecular level, we find that macroH2A is required for the interaction of repeat sequences with the nucleostructural protein lamin B1. Taken together, our results argue that a major function of macroH2A histone variants is to link nucleosome composition to higher-order chromatin architecture.

PARP, transcription and chromatin modeling.

Compaction mode of chromatin and chromatin highly organised structures regulate gene expression. Posttranslational modifications, histone variants and chromatin remodelers modulate the compaction, structure and therefore function of specific regions of chromatin. The generation of poly(ADP-ribose) (PAR) is emerging as one of the key signalling events on sites undergoing chromatin structure modulation. PAR is generated locally in response to stresses. These include genotoxic stress but also differentiation signals, metabolic and hormonal cues. A pictures emerges in which transient PAR formation is essential to orchestrate chromatin remodelling and transcription factors allowing the cell to adapt to alteration in its environment. This review summarizes the diverse factors of ADP-ribosylation in the adaptive regulation of chromatin structure and transcription.

Disruption of Macrodomain Protein SCO6735 Increases Antibiotic Production in Streptomyces coelicolor.

ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and that controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP-ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesize and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor This protein is a member of an uncharacterized subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyze PARP-dependent protein ADP-ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism.