Natarajan Balasubramaniyan

The membrane protein ATPase class I type 8B member 1 signals through protein kinase C zeta to activate the farnesoid X receptor

Prior loss‐of‐function analyses revealed that ATPase class I type 8B member 1 [familial intrahepatic cholestasis 1 (FIC1)] posttranslationally activated the farnesoid X receptor (FXR). Mechanisms underlying this regulation were examined by gain‐of‐function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild‐type and mutated FIC1 expression constructs with a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques. FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR‐dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler disease FIC1 mutants did not activate BSEP, whereas benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent on the presence of the FXR ligand, chenodeoxycholic acid. The effect of FIC1 on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C zeta (PKC ζ) inhibitors (pseudosubstrate or small interfering RNA silencing). Recombinant PKC ζ directly phosphorylated immunoprecipitated FXR. The mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, whereas the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKC ζ in Caco‐2 cells resulted in activation of the human apical sodium‐dependent bile acid transporter promoter. Conclusion: These results demonstrate that FIC1 signals to FXR via PKC ζ. FIC1‐related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. Benign recurrent intrahepatic cholestasis emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity. (HEPATOLOGY 2008;48:1896‐1905.)

Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes

The farnesoid X receptor (FXR) is a ligand (bile acid)-dependent nuclear receptor that regulates target genes involved in every aspect of bile acid homeostasis. Upon binding of ligand, FXR recruits an array of coactivators and associated proteins, some of which have intrinsic enzymatic activity that modify histones or even components of the transcriptional complex. In this study, we show chromatin occupancy by the Set7/9 methyltransferase at the FXR response element (FXRE) and direct methylation of FXR in vivo and in vitro at lysine 206. siRNA depletion of Set7/9 in the Huh-7 liver cell line decreased endogenous mRNAs of the FXR target genes, the short heterodimer partner (SHP) and bile salt export pump (BSEP). Mutation of the methylation site at K206 of FXR to an arginine prevented methylation by Set7/9. A pan-methyllysine antibody recognized the wild-type FXR but not the K206R mutant form. An electromobility shift assay showed that methylation by Set7/9 enhanced binding of FXR/retinoic X receptor-α to the FXRE. Interaction between hinge domain of FXR (containing K206) and Set7/9 was confirmed by coimmunoprecipitation, GST pull down, and mammalian two-hybrid experiments. Set7/9 overexpression in Huh-7 cells significantly enhanced transactivation of the SHP and BSEP promoters in a ligand-dependent fashion by wild-type FXR but not the K206R mutant FXR. A Set7/9 mutant deficient in methyltransferase activity was also not effective in increasing transactivation of the BSEP promoter. These studies demonstrate that posttranslational methylation of FXR by Set7/9 contributes to the transcriptional activation of FXR-target genes.

Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells

ABSTRACT The histone demethylase LSD1, a component of the CoREST (corepressor for element 1-silencing transcription factor) corepressor complex, plays an important role in the downregulation of gene expression during development. However, the activities of LSD1 in mediating short-time-scale gene expression changes have not been well understood. To reveal the mechanisms underlying these two distinct functions of LSD1, we performed genome-wide mapping and cellular localization studies of LSD1 and its dimethylated histone 3 lysine 4 (substrate H3K4me2) in mouse embryonic stem cells (ES cells). Our results showed an extensive overlap between the LSD1 and H3K4me2 genomic regions and a correlation between the genomic levels of LSD1/H3K4me2 and gene expression, including many highly expressed ES cell genes. LSD1 is recruited to the chromatin of cells in the G1/S/G2 phases and is displaced from the chromatin of M-phase cells, suggesting that LSD1 or H3K4me2 alternatively occupies LSD1 genomic regions during cell cycle progression. LSD1 knockdown by RNA interference or its displacement from the chromatin by antineoplastic agents caused an increase in the levels of a subset of LSD1 target genes. Taken together, these results suggest that cell cycle-dependent association and dissociation of LSD1 with chromatin mediates short-time-scale gene expression changes during embryonic stem cell cycle progression.

Identification of Functionally Relevant Residues of the Rat Ileal Apical Sodium-dependent Bile Acid Cotransporter

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na+ ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na+ ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na+.

Adenosine Triphosphate–Binding Cassette Subfamily C Member 2 Is the Major Transporter of the Hepatobiliary Imaging Agent 99mTc-Mebrofenin

The organic anion 99mTc-N-[2-[(3-bromo-2,4,6-trimethylphenyl)-amino]-2-oxoethyl]-N-(carboxymethyl)-glycine (99mTc-mebrofenin) and its analogs are widely used for hepatobiliary imaging. Identification of the mechanisms directing bile canalicular transport of these agents will provide insights into the basis of their hepatic handling for assessing perturbations. Methods: We performed studies in animals, including healthy Fischer 344 rats or rats treated with carbon tetrachloride or intrasplenic cell transplantation and healthy Wistar rats or HsdAMC:TR-Abcc2 mutant rats in Wistar background. Onset of hepatic inflammation was verified by analysis of carbon uptake in Kupffer cells. Hepatic clearance of 99mTc-mebrofenin was studied with dynamic imaging, and fractional retention of peak hepatic mebrofenin activity after 60 min was determined. Changes in the expression of bile canalicular transporters were analyzed by real-time polymerase chain reaction and Western blots. Results: Carbon tetrachloride and cell transplantation produced hepatic inflammation with activation of Kupffer cells, resulting in a rapid decline in the expression of the bile canalicular transporters Abcb4, Abcb11, and Abcc2. Among these transporters, decreased expression of Abcc2 was most prominent, and this decline persisted for 4 wk. Next, we examined 99mTc-mebrofenin excretion in HsdAMC:TR-Abcc2 mutant rats (in which Abcc2 expression is naturally inactivated), compared with their healthy counterparts. In healthy HsdRccHan:WIST rats, only 23% ± 3% of the peak 99mTc-mebrofenin activity was retained after 60 min. By contrast, in HsdAMC:TR-Abcc2 mutant rats, 73% ± 5% of the peak 99mTc-mebrofenin activity was retained (P < 0.001). Moreover, the administration of cyclosporin A markedly inhibited 99mTc-mebrofenin excretion in healthy rats, with no further effect on already impaired 99mTc-mebrofenin excretion in HsdAMC:TR-Abcc2 mutant rats. Hepatic excretion of 99mTc-mebrofenin was largely dependent on Abcc2. This molecular basis of 99mTc-mebrofenin excretion will advance studies of pathophysiologic mechanisms in hepatic Abcc2 pathways.

REGULATION OF DYT1 GENE EXPRESSION BY THE ETS-FAMILY OF TRANSCRIPTION FACTORS

The DYT1 gene encodes for torsinA, a protein with widespread tissue distribution, involved in early onset dystonia (EOD). Numerous studies have focused on torsinA function but no information is available on its transcriptional regulation. We cloned mouse and human 5′‐upstream DYT1 DNA fragments, exhibiting high transcriptional activity, as well as tissue specificity. We identified a proximal minimal DYT1 promoter within −141 bp for mouse and −191 bp for human with respect to the ATG codon. Primer extension analysis indicated multiple transcription start sites. In silico analysis of approximately 500 bp 5′‐upstream DYT1 fragment demonstrated lack of a classical TATA or CAAT box and the presence of a highly conserved direct repeat of two Ets binding cores within −86 bp to −77 bp and −78 bp to −69 bp of the mouse and human DYT1 gene, respectively. A single or a two base nucleotide alteration within the downstream Ets core resulted in approximately 90% (mouse) or 45–60% (human) drop in activity. Interestingly, a 3‐bp distance increase between the two Ets cores dramatically decreased transcriptional activity which was partially restored when the distance was increased up to 10 bp. Ets‐like dominant negatives confirmed the Ets factors as DYT1 transcriptional activators.