Concerted action of the PHD, chromo and motor domains regulates the human chromatin remodelling ATPase CHD4

CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which provide a structural explanation for the regulation of CHD4 activities by intramolecular domain communication. Our results demonstrate functional interdependency between domains within a chromatin remodeller.     

Control of alternative end joining by the chromatin remodeler p400 ATPase

Repair of DNA double-strand breaks occurs in a chromatin context that needs to be modified and remodeled to allow suitable access to the different DNA repair machineries. Of particular importance for the maintenance of genetic stability is the tight control of error-prone pathways, such as the alternative End Joining pathway. Here, we show that the chromatin remodeler p400 ATPase is a brake to the use of alternative End Joining. Using specific intracellular reporter susbstrates we observed that p400 depletion increases the frequency of alternative End Joining events, and generates large deletions following repair of double-strand breaks. This increase of alternative End Joining events is largely dependent on CtIP-mediated resection, indicating that it is probably related to the role of p400 in late steps of homologous recombination. Moreover, p400 depletion leads to the recruitment of poly(ADP) ribose polymerase (PARP) and DNA ligase 3 at DNA double-strand breaks, driving to selective killing by PARP inhibitors. All together these results show that p400 acts as a brake to prevent alternative End Joining-dependent genetic instability and underline its potential value as a clinical marker.     

The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks

DNA damage signaling and repair take place in a chromatin context. Consequently, chromatin-modifying enzymes, including adenosine triphosphate–dependent chromatin remodeling enzymes, play an important role in the management of DNA double-strand breaks (DSBs). Here, we show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, although p400 is not required for DNA damage signaling, DNA DSB repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair, and survival after DNA damage. Strikingly, p400 and Rad51 are present in the same complex and both favor chromatin remodeling around DSBs. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodeling enzyme involved in chromatin decompaction around DNA DSBs.     

The FUN30 chromatin remodeler, Fft3, protects centromeric and subtelomeric domains from euchromatin formation

The chromosomes of eukaryotes are organized into structurally and functionally discrete domains. This implies the presence of insulator elements that separate adjacent domains, allowing them to maintain different chromatin structures. We show that the Fun30 chromatin remodeler, Fft3, is essential for maintaining a proper chromatin structure at centromeres and subtelomeres. Fft3 is localized to insulator elements and inhibits euchromatin assembly in silent chromatin domains. In its absence, euchromatic histone modifications and histone variants invade centromeres and subtelomeres, causing a mis-regulation of gene expression and severe chromosome segregation defects. Our data strongly suggest that Fft3 controls the identity of chromatin domains by protecting these regions from euchromatin assembly.     

The regulatory tandem domains of human phosphodiesterases 1 and 4 regulate a cyanobacterial adenylyl cyclase

Human phosphodiesterase 1 is regulated by a tandem of N-terminal calmodulin/Ca(2+)-binding domains. We grafted the tandems from hPDE1A3 and -B1 onto the cyanobacterial adenylyl cyclase CyaB1 thus replacing an intrinsic tandem GAF-domain. Cyclase activity was stimulated by Ca(2+)/calmodulin 1.9 to 4.4-fold, i.e. similarly as reported for hPDE1 regulation. hPDE4 long isoforms are activated by phosphorylation of a serine located in a conserved RRESF motif in a tandem of N-terminal upstream-conserved regions (UCR). We grafted the UCR tandems from hPDE4A4, -B1, and -D3 onto the CyaB1 cyclase as a reporter enzyme. Activity was enhanced 1.4 to 4.5-fold by respective phosphomimetic (S/D) point mutations. Similarly, cyclase activity was increased 2.5-fold by phosphorylation of the chimera with the PDE4D3 UCR tandem by cAMP-dependent protein kinase. We propose a common mechanism of activation in mammalian phosphodiesterases containing N-terminal tandem regulatory domains. A downstream region is suggested to alternate between random and ordered conformations and to enable switching between inactive, the catalytic domain occluding PDE homodimers and active monomeric PDE catalytic domains.     

The chromatin remodeler RSC prevents ectopic CENP-A propagation into pericentromeric heterochromatin at the chromatin boundary

Centromeres of most eukaryotes consist of two distinct chromatin domains: a kinetochore domain, identified by the histone H3 variant, CENP-A, and a heterochromatic domain. How these two domains are separated is unclear. Here, we show that, in Schizosaccharomyces pombe, mutation of the chromatin remodeler RSC induced CENP-A^Cnp1 misloading at pericentromeric heterochromatin, resulting in the mis-assembly of kinetochore proteins and a defect in chromosome segregation. We find that RSC functions at the kinetochore boundary to prevent CENP-A^Cnp1 from spreading into neighbouring heterochromatin, where deacetylated histones provide an ideal environment for the spread of CENP-A^Cnp1. In addition, we show that RSC decompacts the chromatin structure at this boundary, and propose that this RSC-directed chromatin decompaction prevents mis-propagation of CENP-A^Cnp1 into pericentromeric heterochromatin. Our study provides an insight into how the distribution of distinct chromatin domains is established and maintained.     

The CHD3 remodeler PICKLE promotes trimethylation of histone H3 lysine 27

CHD3 proteins are ATP-dependent chromatin remodelers that contribute to repression of developmentally regulated genes in both animal and plant systems. In animals, this repression has been linked to a multiple subunit complex, Mi-2/NuRD, whose constituents include a CHD3 protein, a histone deacetylase, and a methyl-CpG-binding domain protein. In Arabidopsis, PICKLE (PKL) codes for a CHD3 protein that acts during germination to repress expression of seed-associated genes. Repression of seed-associated traits is promoted in pkl seedlings by the plant growth regulator gibberellin (GA). We undertook a microarray analysis to determine how PKL and GA act to promote the transition from seed to seedling. We found that PKL and GA act in separate pathways to repress expression of seed-specific genes. Comparison of genomic datasets revealed that PKL-dependent genes are enriched for trimethylation of histone H3 lysine 27 (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation studies demonstrate that PKL promotes H3K27me3 in both germinating seedlings and in adult plants but do not identify a connection between PKL-dependent expression and acetylation levels. Taken together, our analyses illuminate a new pathway by which CHD3 remodelers contribute to repression in eukaryotes.     

The chromatin remodeler BRAHMA recruits HISTONE DEACETYLASE6 to regulate root growth inhibition in response to phosphate starvation in Arabidopsis

Plasticity in root system architecture(RSA) allows plants to adapt to changing nutritional status in the soil.Phosphorus availability is a major determinant of crop yield,and RSA remodeling is critical to increasing the efficiency of phosphorus acquisition.Although substantial progress has been made in understanding the signaling mechanism driving phosphate starvation responses in plants,whether and how epigenetic regulatory mechanisms contribute is poorly understood.Here,we report that the Swit...     

ATPase activity of nucleotide binding domains of human MDR3 in the context of MDR1

Although human MDR1 and MDR3 share 86% similarity in their amino acid sequences and are predicted to share conserved domains for drug recognition, their physiological transport substrates are quite different: MDR1 transports xenobiotics and confers multidrug resistance, while MDR3 exports phosphatidylcholine into bile. Although MDR1 shows high ATPase activity, attempts to demonstrate the ATPase activity of human MDR3 have not succeeded. Therefore, it is possible that the difference in the functions of these proteins is caused by their different ATPase activities. To test this hypothesis, a chimera protein containing the transmembrane domains (TMDs) of MDR1 and the nucleotide binding domains (NBDs) of MDR3 was constructed and analyzed. The chimera protein was expressed on the plasma membrane and conferred resistance against vinblastine and paclitaxel, indicating that MDR3 NBDs can support drug transport. Vanadate-induced ADP trapping of MDR3 NBDs in the chimera protein was stimulated by verapamil as was MDR1 NBDs. The purified chimera protein showed drug-stimulated ATPase activity like MDR1, while its V_max was more than 10-times lower than MDR1. These results demonstrate that the low ATPase activity of human MDR3 cannot account for the difference in the functions of these proteins, and furthermore, that TMDs determine the features of NBDs. To our knowledge, this is the first study analyzing the features of human MDR3 NBDs.     

The chromatin remodeler ISWI regulates the cellular response to hypoxia: role of FIH

The hypoxia-inducible factor (HIF) is a master regulator of the cellular response to hypoxia. Its levels and activity are controlled by dioxygenases called prolyl-hydroxylases and factor inhibiting HIF (FIH). To activate genes, HIF has to access sequences in DNA that are integrated in chromatin. It is known that the chromatin-remodeling complex switch/sucrose nonfermentable (SWI/SNF) is essential for HIF activity. However, no additional information exists about the role of other chromatin-remodeling enzymes in hypoxia. Here we describe the role of imitation switch (ISWI) in the cellular response to hypoxia. We find that unlike SWI/SNF, ISWI depletion enhances HIF activity without altering its levels. Furthermore, ISWI knockdown only alters a subset of HIF target genes. Mechanistically, we find that ISWI is required for full expression of FIH mRNA and protein levels by changing RNA polymerase II loading to the FIH promoter. Of interest, exogenous FIH can rescue the ISWI-mediated upregulation of CA9 but not BNIP3, suggesting that FIH-independent mechanisms are also involved. Of importance, ISWI depletion alters the cellular response to hypoxia by reducing autophagy and increasing apoptosis. These results demonstrate a novel role for ISWI as a survival factor during the cellular response to hypoxia.     

Expansion of the ISWI chromatin remodeler family with new active complexes

ISWI chromatin remodelers mobilize nucleosomes to control DNA accessibility. Complexes isolated to date pair one of six regulatory subunits with one of two highly similar ATPases. However, we find that each endogenously expressed ATPase co‐purifies with every regulatory subunit, substantially increasing the diversity of ISWI complexes, and we additionally identify BAZ2B as a novel, seventh regulatory subunit. Through reconstitution of catalytically active human ISWI complexes, we demonstrate that the new interactions described here are stable and direct. Finally, we profile the nucleosome remodeling functions of the now expanded family of ISWI chromatin remodelers. By revealing the combinatorial nature of ISWI complexes, we provide a basis for better understanding ISWI function in normal settings and disease.     

Chromosomal elements regulate gene activity and chromatin structure of the human serpin gene cluster at 14q32.1

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 contains a number of genes that are specifically expressed in hepatic cells. Cell-specific enhancers have been identified in several of these genes, but elements involved in locus-wide gene and chromatin control have yet to be defined. To identify regulatory elements in this region, we prepared a series of mutant chromosomal alleles by homologous recombination and transferred the specifically modified human chromosomes to hepatic cells for functional tests. We report that deletion of an 8-kb DNA segment upstream of the human alpha1-antitrypsin gene yields a mutant serpin allele that fails to be activated in hepatic cells. Within this region, a 2.3-kb DNA segment between kb -8.1 and -5.8 contains a previously unrecognized control region that is required not only for serpin gene activation but also for chromatin remodeling of the entire locus.     

Multivalent Recognition of Histone Tails by the PHD Fingers of CHD5

The chromodomain, helicase, DNA-binding protein 5 (CHD5) is a chromatin remodeling enzyme which is implicated in tumor suppression. In this study, we demonstrate the ability of the CHD5 PHD fingers to specifically recognize the unmodified N-terminus of histone H3. We use two distinct modified peptide-library platforms (beads and glass slides) to determine the detailed histone binding preferences of PHD(1) and PHD(2) alone and the tandem PHD(1-2) construct. Both domains displayed similar binding preferences for histone H3, where modification (e.g., methylation, acetylation, and phosphorylation) at H3R2, H3K4, H3T3, H3T6, and H3S10 disrupts high-affinity binding, and the three most N-terminal amino acids (ART) are crucial for binding. The tandem CHD5-PHD(1-2) displayed similar preferences to those displayed by each PHD finger alone. Using NMR, surface plasmon resonance, and two novel biochemical assays, we demonstrate that CHD5-PHD(1-2) simultaneously engages two H3 N-termini and results in a 4-11-fold increase in affinity compared with either PHD finger alone. These studies provide biochemical evidence for the utility of tandem PHD fingers to recruit protein complexes at targeted genomic loci and provide the framework for understanding how multiple chromatin-binding modules function to interpret the combinatorial PTM capacity written in chromatin.