Chemical modulation of histone deacetylase (HDAC) activity by HDAC inhibitors (HDACi) is an increasingly important approach to modify the etiology of human disease. Loss-of-function diseases arise as a consequence of protein misfolding and degradation leading to system failures. The ΔF508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) results in the absence of the cell surface chloride channel and a loss of airway hydration, leading to premature lung failure and reduced lifespan responsible for cystic fibrosis (CF). We now show that the HDACi suberoylanilide hydroxamic acid (SAHA) restores surface channel activity in human primary airway epithelia to levels that are 28% of wild-type CFTR. Biological silencing of all known class I and II HDACs reveals that HDAC7 plays a central role in restoration of ΔF508 function. We suggest that the tunable capacity of HDACs can be manipulated by chemical biology to counter the onset of CF and other human misfolding disorders.
Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPβ binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.
Histone 3 lysine 9 (H3K9) demethylase JMJD1A regulates β-adrenergic-induced systemic metabolism and body weight control. Here we show that JMJD1A is phosphorylated at S265 by protein kinase A (PKA), and this is pivotal to activate the β1-adrenergic receptor gene (Adrb1) and downstream targets including Ucp1 in brown adipocytes (BATs). Phosphorylation of JMJD1A by PKA increases its interaction with the SWI/SNF nucleosome remodelling complex and DNA-bound PPARγ. This complex confers β-adrenergic-induced rapid JMJD1A recruitment to target sites and facilitates long-range chromatin interactions and target gene activation. This rapid gene induction is dependent on S265 phosphorylation but not on demethylation activity. Our results show that JMJD1A has two important roles in regulating hormone-stimulated chromatin dynamics that modulate thermogenesis in BATs. In one role, JMJD1A is recruited to target sites and functions as a cAMP-responsive scaffold that facilitates long-range chromatin interactions, and in the second role, JMJD1A demethylates H3K9 di-methylation.
CLC-K1 is a kidney-specific chloride channel that mediates transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) in the inner medulla. Transport of NaCl in the tAL is thought to be a component of urinary concentration in a passive model of the countercurrent multiplication system, but there has been no direct evidence that CLC-K1 is involved in urine concentration. To analyse the physiological function of CLC-K1 in vivo, we generated mice lacking CLC-K1 by targeted gene disruption. Clcnk1-/- mice were physically normal appearance, but produced approximately five times more urine than Clcnk1+/- and Clcnk1+/+ mice. After 24 hours of water deprivation, Clcnk1-/- mice were severely dehydrated and lethargic, with a decrease of approximately 27% in body weight. Intraperitoneal injection of the V2 agonist 1-deamino-8-D-arginine vasopressin (dDAVP) induced a threefold increase in urine osmolarity in Clcnk1+/- and Clcnk1+/+ mice, whereas only a minimal increase was seen in Clcnk1-/- mice, indicating nephrogenic diabetes insipidus. After in vitro perfusion of the tAL, the lumen-to-bath chloride gradient did not produce a diffusion potential in Clcnk1-/- mice in contrast to Clcnk1+/+ and Clcnk1+/- mice. These results establish that CLC-K1 has a role in urine concentration, and that the countercurrent system in the inner medulla is involved in the generation and maintenance of hypertonic medullary interstitium.
The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency.
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