Chromodomains are modules implicated in the recognition of lysine-methylated histone tails and nucleic acids. CHD (for chromo-ATPase/helicase-DNA-binding) proteins regulate ATP-dependent nucleosome assembly and mobilization through their conserved double chromodomains and SWI2/SNF2 helicase/ATPase domain. The Drosophila CHD1 localizes to the interbands and puffs of the polytene chromosomes, which are classic sites of transcriptional activity. Other CHD isoforms (CHD3/4 or Mi-2) are important for nucleosome remodelling in histone deacetylase complexes. Deletion of chromodomains impairs nucleosome binding and remodelling by CHD proteins. Here we describe the structure of the tandem arrangement of the human CHD1 chromodomains, and its interactions with histone tails. Unlike HP1 and Polycomb proteins that use single chromodomains to bind to their respective methylated histone H3 tails, the two chromodomains of CHD1 cooperate to interact with one methylated H3 tail. We show that the human CHD1 double chromodomains target the lysine 4-methylated histone H3 tail (H3K4me), a hallmark of active chromatin. Methylammonium recognition involves two aromatic residues, not the three-residue aromatic cage used by chromodomains of HP1 and Polycomb proteins. Furthermore, unique inserts within chromodomain 1 of CHD1 block the expected site of H3 tail binding seen in HP1 and Polycomb, instead directing H3 binding to a groove at the inter-chromodomain junction.
Double chromodomains occur in CHD proteins which are ATP-dependent chromatin remodeling factors implicated in RNA polymerase II transcription regulation. Biochemical studies suggest important differences in the histone H3 tail binding of different CHD chromodomains. In human and Drosophila, CHD1 double chromodomains bind lysine 4-methylated histone H3 tail which is a hallmark of transcriptionally active chromatin in all eukaryotes. Here, we present the crystal structure of the yeast CHD1 double chromodomains, and pinpoint its differences with that of the human CHD1 double chromodomains. The most conserved residues in these double chromodomains are the two chromoboxes that orient adjacently. Only a subset of CHD chromoboxes can form an aromatic cage for methyllysine binding, and methyllysine binding requires correctly oriented inserts. These factors preclude yeast CHD1 double chromodomains to interact with the histone H3 tail. Despite great sequence similarity between the human CHD1 and CHD2 chromodomains, variation within an insert likely prevents CHD2 double chromodomains to bind lysine 4-methylated histone H3 tail as efficiently as in CHD1. By using the available structural and biochemical data we highlight the evolutionary specialization of CHD double chromodomains, and provide insights about their targeting capacities.
Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover—impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr−/−) mouse model. In the murine calvarial organ culture assay, Cftr−/− calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr−/− compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr−/− compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr−/− murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr−/− murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.
Rck2p is a Ser/Thr kinase that binds to, and is activated by, Hog1p. Expression of the MAP kinase kinase Pbs2pDD from a GAL1-driven plasmid hyperactivates the HOG MAP kinase pathway, and leads to cessation of growth. This toxic effect is reduced by deletion of RCK2. We studied the structural and functional basis for the role of Rck2p in mediating the growth arrest phenotype associated with overexpression of Pbs2pDD. Rck2p kinase activity is required for the effect, because Rck2p(Delta487-610), as well as full-length Rck2p, is toxic with Pbs2pDD, but kinase-defective versions of either protein with a K201R mutation are not. Thus, the C-terminal portion of Rck2p is not required provided the protein is activated by removal of the autoinhibitory domain. Relief of inhibition in Rck2p normally requires phosphorylation by Hog1p, and Rck2p contains a putative MAP kinase docking site (TILQR589R590KKVQ) in its C-terminal segment. The Rck2p double mutant R589A/R590A expressed from a centromeric plasmid did not detectably bind Hog1p-GFP and was functionally inactive in mediating the toxic effect of Pbs2pDD, equivalent to an RCK2 deletion. However, overexpression of Rck2p R589A/R590A from a multicopy plasmid restored function. In contrast, RCK2-K201R acted as a multicopy suppressor of PBS2DD, markedly reducing its toxicity. This suppressor activity required the K201R mutation, and the effect was largely lost when the docking site was mutated, suggesting suppression by inhibition of Hog1p functions. We also studied the effect of replacing the predicted T379 and established S520 phosphorylation sites in Rck2p by glutamic acid. Surprisingly, the T379E mutant markedly reduced Pbs2pDD toxicity, and toxicity was only partially rescued by S520E. Rck2 T379E was sufficiently inactive in an rck2Delta strain to allow some cells to survive PBS2DD toxicity even when overexpressed. The significance of these findings for our understanding of Rck2p function is discussed.
Endothelin-1 (ET-1) is a potent vasoconstrictor that also stimulates cells in the osteoblast lineage by binding to the endothelin A receptor (ETAR). ET-1 ligand is widely secreted, particularly by the vasculature. However, the contributions of ETAR signaling to adult bone homeostasis had not been defined. ETAR was inactivated in osteoblasts by crossing ETAR-floxed and osteocalcin-Cre mice. Histomorphometric analyses were performed on 4, 8 and 12 week-old osteoblast-targeted ETAR knockout (KO) and wild type (WT) male and female mice. Tibial trabecular bone volume was significantly lower from 12 weeks in KO vs. WT mice in both males and females. Bone formation rate, osteoblast density and in vitro osteoblast differentiation were reduced by targeted inactivation of ETAR. A separate longitudinal analysis was performed between 8 and 64 weeks, to examine the effect of aging and castration on bone metabolism in ETAR KO mice. Hypogonadism did not change the rate of bone accrual in WT or KO females. However, eugonadal KO males had a significantly larger increase in tibial and femoral bone acquisition compared to WT. Male mice castrated at 8 weeks of age showed the reverse: KO mice had reduced rates of tibial and femoral BMD acquisition compared to WT. In vitro, ET-1 increased osteoblast proliferation, survival and differentiation. Dihydrotestosterone also increased osteoblast differentiation using a mechanism distinct from the actions of ET-1. These results demonstrate that endothelin signaling in osteoblasts is an important regulator of postnatal trabecular bone remodeling and a modulator of androgen effects on bone.
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