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Tissue fibrosis is a major cause of organ dysfunction during chronic diseases and aging. A critical step in this process is transforming growth factor β1 (TGF-β1)-mediated transformation of fibroblasts into myofibroblasts, cells capable of synthesizing extracellular matrix. Here, we show that SIRT3 controls transformation of fibroblasts into myofibroblasts via suppressing the profibrotic TGF-β1 signaling. We found that Sirt3 knockout (KO) mice with age develop tissue fibrosis of multiple organs, including heart, liver, kidney, and lungs but not whole-body SIRT3-overexpressing mice. SIRT3 deficiency caused induction of TGF-β1 expression and hyperacetylation of glycogen synthase kinase 3β (GSK3β) at residue K15, which negatively regulated GSK3β activity to phosphorylate the substrates Smad3 and β-catenin. Reduced phosphorylation led to stabilization and activation of these transcription factors regulating expression of the profibrotic genes. SIRT3 deacetylated and activated GSK3β and thereby blocked TGF-β1 signaling and tissue fibrosis. These data reveal a new role of SIRT3 to negatively regulate aging-associated tissue fibrosis and discloses a novel phosphorylation-independent mechanism controlling the catalytic activity of GSK3β.

The human ISWI-containing factor RSF (for remodeling and spacing factor) is composed of two subunits: the ATPase hSNF2H and p325 (Rsf-1), a protein encoded by a novel human gene. We previously showed that RSF mediates nucleosome deposition and generates regularly spaced nucleosome arrays. Here we report the characterization of the largest subunit of RSF, Rsf-1. We found that Rsf-1 is a highly acidic protein containing a plant homology domain. The present study includes the cloning of Rsf-1, the preparation of recombinant RSF, and the dissection of the role of each subunit in the chromatin assembly reaction. The sequence of the gene for Rsf-1 includes a recently characterized cDNA, HBXAP; postulated to be involved in the transcriptional regulation of the hepatitis B virus. HBXAP actually contains a 252-amino-acid truncation of the amino terminus of Rsf-1. Finally, comparison of HBXAP and Rsf-1 properties shows that they are functionally different.

Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity. The nucleolus in eukaryotic cells carries out most of the important reactions in ribosome biogenesis, including rDNA (rRNA genes) transcription, rRNA processing, and preribosome assembly (34, 47, 49, 50). Elements of nucleolar architecture mediate a tight temporal and topological orchestration of these processes. In higher eukaryotic cells, nucleoli exhibit a common organization consisting of three ultrastructurally distinct regions, the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). The main body of the nucleolus is made up of the GC, embedded in this granular mass are several islets of rounded structures, FCs, each surrounded by a compact layer of the DFC. Tandemly repeated rRNA genes are clustered mostly in the FCs (42, 57), with transcription occurring largely at the boundary between the FC and the DFC (13, 20, 42, 58). Nascent rRNA transcripts are processed in the DFC (25, 38, 59). Some processing steps may also occur in the GC, together with the assembly of the mature rRNA and ribosomal proteins into preribosomal subunits (42, 47). In addition to rDNA, rRNA, and ribosomal proteins, the nucleolus harbors a large number of nonribosomal proteins and small nucleolar RNAs, which mediate the transcriptional and posttranscriptional reactions of ribosome biogenesis (49, 54). Many nucleolar proteins locate in particular nucleolar regions, which may reflect functional compart