The ATP-dependent Chromatin Remodeling Enzyme Fun30 Represses Transcription by Sliding Promoter-proximal Nucleosomes

Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.

The human ACF chromatin-remodeling complex (hACF) contains the ATPase motor protein SNF2h and the non-catalytic hACF1 subunit. Here, we have compared the ability of SNF2h and a reconstituted hACF complex containing both SNF2h and hACF1 to remodel a series of nucleosomes containing different lengths of DNA overhang. Both SNF2h and hACF functioned in a manner consistent with sliding a canonical nucleosome. However, the non-catalytic subunit, hACF1, altered the remodeling properties of SNF2h by changing the nature of the requirement for a DNA overhang in the nucleosomal substrate and altering the DNA accessibility profile of the remodeled products. Surprisingly, addition of hACF1 to SNF2h increased the amount of DNA overhang needed to observe measurable amounts of DNA accessibility, but decreased the amount of overhang needed for a measurable binding interaction. We propose that these hACF1 functions might contribute to making the hACF complex more efficient at nucleosome spacing compared with SNF2h. In contrast, the SWI/SNF complex and its ATPase subunit BRG1 generated DNA accessibility profiles that were similar to each other, but different significantly from those of hACF and SNF2h. Thus, we observed divergent remodeling behaviors in these two remodeling families and found that the manner in which hACF1 alters the remodeling behavior of the ATPase is not shared by SWI/SNF subunits.

The paradigm of activation via ordered recruitment has evolved into a complicated picture as the influence of coactivators and chromatin structures on gene regulation becomes understood. We present here a comprehensive study of many elements of activation of ADH2 and FBP1, two glucose-regulated genes. We identify SWI/SNF as the major chromatin-remodeling complex at these genes, whereas SAGA (Spt-Ada-Gcn5-acetyltransferase complex) is required for stable recruitment of other coactivators. Mediator plays a crucial role in expression of both genes but does not affect chromatin remodeling. We found that Adr1 bound unaided by coactivators to ADH2, but Cat8 binding depended on coactivators at FBP1. Taken together, our results suggest that commonly regulated genes share many aspects of activation, but that gene-specific regulators or elements of promoter architecture may account for small differences in the mechanism of activation. Finally, we found that activator overexpression can compensate for the loss of SWI/SNF but not for the loss of SAGA.

Role of Nucleosomal Occupancy in the Epigenetic Silencing of the MLH1 CpG Island

SWI/SNF Has Intrinsic Nucleosome Disassembly Activity that Is Dependent on Adjacent Nucleosomes

In budding yeast and human cells, ING (inhibitor of growth) tumor suppressor proteins play important roles in response to DNA damage by modulating chromatin structure through collaborating with histone acetyltransferase or histone deacetylase complexes. However, the biological functions of ING family proteins in fission yeast are poorly defined. Here, we report that Png1p, a fission yeast ING homolog protein, is required for cell growth under normal and DNA-damaged conditions. Png1p was further confirmed to regulate histone H4 acetylation through collaboration with the MYST family histone acetyltransferase 1 (Mst1). Additionally, both fission yeast PNG1 and MST1 can functionally complement their budding yeast correspondence homologs YNG2 and ESA1, respectively. These results suggest that ING proteins in fission yeast might also conserve function, similar to ING proteins in budding yeast and human cells. We also showed that decreased acetylation in Deltapng1 cells resulted in genome-wide down-regulation of 756 open reading frames, including the central DNA repair gene RAD22. Overexpression of RAD22 partially rescued the png1 mutant phenotype under both normal and DNA-damaged conditions. Furthermore, decreased expression of RAD22 in Deltapng1 cells was confirmed to be caused by decreased H4 acetylation at its promoter. Altogether, these results indicate that Png1p is required for histone H4 acetylation and functions upstream of RAD22 in the DNA damage response pathway.

Nucleosome Transactions on the Promoters of the YeastGAL and PHO Genes

The influence of chromatin structure on DNA metabolic processes, including DNA replication and repair, has been a matter of intensive studies in recent years. Although the human mismatch repair (MMR) reaction has been reconstituted using purified proteins, the influence of chromatin structure on human MMR is unknown. This study examines the interaction between human MutSalpha and a mismatch located within a nucleosome or between two nucleosomes. The results show that, whereas MutSalpha specifically recognizes both types of nucleosomal heteroduplexes, the protein bound the mismatch within a nucleosome with much lower efficiency than a naked heteroduplex or a heterology free of histone proteins but between two nucleosomes. Additionally, MutSalpha displays reduced ATPase- and ADP-binding activity when interacting with nucleosomal heteroduplexes. Interestingly, nucleosomes block ATP-induced MutSalpha sliding along the DNA helix when the mismatch is in between two nucleosomes. These findings suggest that nucleosomes may inhibit MMR in eukaryotic cells. The implications of these findings for our understanding of eukaryotic MMR are discussed.

Changes in chromatin architecture induced by epigenetic mechanisms are essential for normal cellular processes such as gene expression, DNA repair, and cellular division. Compact chromatin presents a barrier to these processes and is highly regulated by epigenetic markers binding to components of the nucleosome. Histone modifications directly influence chromatin dynamics and facilitate recruitment of additional factors such as chromatin remodelers and histone chaperones. One member of this last class of factors, FACT (facilitates chromatin transcription), is categorized as a histone chaperone critical for nucleosome reorganization during replication, transcription, and DNA repair. Significant discoveries regarding the role of histone chaperones and specifically FACT have come over the past dozen years from a number of independent laboratories. Here, we review the structural and biophysical basis for FACT-mediated nucleosome reorganization and discuss up-to-date models for FACT function.

Brahma (Brm) and Brahma-related gene-1 (Brg1) ATPases share similarities in structure and function, but their presence in human SWI/SNF chromatin remodeling complexes is mutually exclusive. Although Brm and Brg1 can compensate for each other, it is possible that Brm and Brg1 have their unique properties to differentially regulate gene expression in vivo. To explore this, we examined the requirement of Brm and Brg1 for p53-dependent transcription, especially p53-mediated induction of p21 and MDM2, using cell lines in which Brm or Brg1 could be inducibly knocked down. We found that Brg1, but not Brm, is required for p21 induction in MCF7 cells. However, in Brg1-deficient H1299 cells, Brm is also required for p21 induction. Likewise, Brm is necessary for induction of p21 in MCF7 cells in which Brg1 is stably knocked down. In contrast, Brg1 has little, if any, effect on p53-mediated induction of MDM2 in cells that have Brm and vice versa. In addition, we demonstrated that the impaired induction of p21 upon Brg1 knockdown is at least in part due to decreased p53 binding to the p21 promoter. Taken together, we provided evidence that Brg1 is preferentially recruited by p53 for inducing a subset of target genes through chromatin remodeling. Thus, we hypothesize that the potential tumor suppressor function for Brg1 is mediated in part through the p53 pathway.

Higher-order orchestration of hematopoiesis: Is cohesin a new player?

The mammalian SWI/SNF chromatin remodeling complex is a key player in multiple chromatin transactions. Core subunits of this complex, including the ATPase, Brg-1, and various Brg-1-associated factors (BAFs), work in concert to maintain a functional remodeling complex. This intra-complex regulation is supervised by protein-protein interactions, as stoichiometric levels of BAF proteins are maintained by proteasomal degradation. We show that the mechanism of BAF155-mediated stabilization of BAF57 involves blocking its ubiquitination by preventing interaction with TRIP12, an E3 ubiquitin ligase. Consequently, as opposed to complexed BAF57, whose principal lysines are unavailable for ubiquitination, uncomplexed BAF57 can be freely ubiquitinated and degraded by the proteasome. Additionally, a BAF57 mutant, which contains no lysine residues, was found to retain its ability to be stabilized by interaction with BAF155, suggesting that in addition to the ubiquitin-dependent mechanism of BAF57 degradation, there exists a ubiquitin-independent mechanism that may involve the direct interaction of BAF57 with the proteasome. We propose that this regulatory mechanism exists to ensure functional fidelity of the complex and prevent the accumulation of uncomplexed proteins, which may disrupt the normal activity of the complex.

Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.

hormone response element peroxisome proliferator-activated receptor thyroid hormone receptor estrogen receptor ligand binding domain nuclear receptor corepressor silencing mediator of retinoic acid and thyroid hormone receptor imitation SWI cAMP response element-binding protein CREB-binding protein histone acetyltransferase mitogen-activated protein histone deacetylase steroid receptor coactivator RAR interacting protein glucocorticoid receptor interacting protein T3R receptor associated protein vitamin receptor D interacting protein Members of the nuclear receptor superfamily directly activate or repress target genes by binding to hormone response elements (HREs)1 in promoter or enhancer regions, and by binding to other DNA sequence-specific activators and can inhibit the transcriptional activities of other classes of transcription factors by transrepression. Hormone response elements provide specificity to receptor homodimer heterodimer binding (reviewed in Ref. 2Bourguet W. Germain P. Gronemeyer H. Trends Pharm. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Nuclear receptor functions are directed by specific activation domains, referred to as activation function 1 (AF-1), which resides in the N terminus, and activation function 2 (AF-2), which resides in the C-terminal ligand binding domain (LBD) (reviewed in Ref. 1Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Regulation of gene transcription by nuclear receptors requires the recruitment of proteins characterized as coregulators, with ligand-dependent exchange of corepressors for coactivators serving as the basic mechanism for switching gene repression to activation. In this review, we discuss biochemical and genetic studies suggesting that coregulatory complexes are differentially utilized in both a cell- and promoter-specific fashion to activate or repress gene transcription. These coregulatory components, themselves targets of diverse intracellular signaling pathways, provide a combinatorial code for tissue- and gene-specific responses, utilizing both enzymatic and platform assembly functions to mediate the actions of nuclear receptor genetic programs critical for developmental and homeostatic processes in metazoan organisms. A diverse group of proteins have emerged as potential coactivators for nuclear receptors. Ligand-dependent recruitment of coactivators is dependent on AF-2, which consists of a short conserved helical sequence within the C terminus of the LBD (2Bourguet W. Germain P. Gronemeyer H. Trends Pharm. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Biochemical and expression cloning approaches have been used to identify a large number of factors that interact with nuclear receptors in either a ligand-independent or a ligand-dependent manner and are often components of large multiprotein complexes. Many of these factors are capable of potentiating nuclear receptor activity in transient cotransfection assays. In addition, a distinct set of coactivators is associated with the AF-1 domain. As the number of potential coregulators clearly exceeds the capacity for direct interaction by a single receptor, the most plausible hypothesis is that transcriptional activation by nuclear receptors involves the actions of multiple factors. These factors act in a sequential and/or combinatorial manner to reorganize chromatin templates and to modify and recruit basal factors and RNA polymerase II (3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). As chromatinized transcription units are “repressed” compared with naked DNA, a critical aspect of gene activation involves nucleosomal remodeling (reviewed in Refs. 3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Two general classes of chromatin remodeling factors that appear to play critical roles in transcriptional activation by nuclear receptors have been identified. These are ATP-dependent nucleosome remodeling complexes and factors that contain histone acetyltransferase activity. The yeast SWI·SNF complex facilitates the binding of sequence-specific transcription factors to nucleosomal DNA and can cause local changes in chromatin structure in an ATP-dependent manner (3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 6Pazin M.J. Kadonaga J.T. Cell. 1997; 88: 737-740Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar, 8Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 9Ogryzko V.V. Kotani T. Zhang R.L. Howard S.T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 10Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar, 12Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (897) Google Scholar). Mammalian homologues of Drosophila SWI2/SNF2 such as BRG1/hBrm function as components of large multiprotein complexes. Transfection of ATPase-defective alleles of either Brg1 orhBrm into several mammalian cell lines leads to a significant decrease in the ability of several nuclear receptors to activate transcription (3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 6Pazin M.J. Kadonaga J.T. Cell. 1997; 88: 737-740Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Remodeling complexes containing ISWI (imitation SWI) may also be involved in nuclear receptor function (7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar, 8Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 9Ogryzko V.V. Kotani T. Zhang R.L. Howard S.T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 10Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar). Rates of gene transcription roughly correlate with the degree of histone acetylation, with hyperacetylated regions of the genome appearing to be more actively transcribed than hypoacetylated regions (reviewed in Ref. 7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). The specific recruitment of a complex with histone acetyltransferase activity to a promoter may play a critical role in overcoming repressive effects of chromatin structure on transcription (4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 6Pazin M.J. Kadonaga J.T. Cell. 1997; 88: 737-740Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). This concept was further supported by the subsequent finding that the mammalian Gcn5 orthologues, including p/CAF, CREB-binding protein (CBP), adenovirus E1A-binding protein p300, and TAFII250, each possess intrinsic histone acetyltransferase (HAT) activity (7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar, 8Mizzen C.A. Yang X.-J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 9Ogryzko V.V. Kotani T. Zhang R.L. Howard S.T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 10Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar). Conversely, the discovery that a mammalian histone deacetylase (HDAC) was a homologue of the yeast corepressor, RPD3 (13Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1569) Google Scholar), gave rise to the hypothesis that regulated activation events might involve the exchange of complexes containing histone deacetylase functions with those containing histone acetyltransferase activity (Fig. 1). It appears that in most cases the acetyltransferases are not directly recruited to nuclear receptors but associate with other coactivators that exhibit higher affinity for the liganded receptor. The acetyltransferase functions of factors such as CBP/p300 are directly required for enhanced transcription on chromatinized templates (14Kraus W. Manning E. Kadonaga J. Mol. Cell Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (203) Google Scholar). A large number of proteins that are recruited in a ligand-dependent fashion have the capacity to enhance transcriptional activation by transient transfection. Several insights into the mechanisms by which coactivator complexes are recruited to nuclear receptors in a ligand-dependent manner have been provided by the initial identification of the p160 family of nuclear receptor coactivators, referred to as SRC-1/NCOA1, TIF2/GRIP1, and p/CIP/A1B1/ACTR/RAC/TRAM-1 (reviewed in Ref. 15McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1669) Google Scholar). The p160 factors consist of three members that exhibit a common domain structure, illustrated in Fig. 1. The central conserved domain mediates ligand-dependent interactions with the nuclear receptor LBD, whereas the conserved C-terminal transcriptional activation domains mediate interactions with either CBP/p300 or protein-arginine methyltransferase (16Chen D. Ma H. Hong H. Koh S.S. Huang S.-M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2176Crossref PubMed Scopus (1019) Google Scholar, 17Koh S. Chen D. Lee Y. Stallcup M. J. Biol. Chem. 2001; 276: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Based on the presence of three regulatory domains, members of the p160 family have been suggested to function as coactivators, at least in part, by serving as adapter molecules that recruit CBP and/or p300 complexes to promoter-bound nuclear receptors in a ligand-dependent manner (18Kurokawa R. Kalafus D. Ogliastro M.-H. Kioussi C. Xu L. Torchia J. Rosenfeld M.G. Glass C.K. Science. 1998; 279: 700-703Crossref PubMed Scopus (199) Google Scholar, 19Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1112) Google Scholar). Biochemical studies have also demonstrated strong ligand-dependent interactions between nuclear receptors and p140 factors, probably representing the coregulator RIP140, which results in a reproductive defect in female mice on gene deletion (20White R. Leonaardsson G. Roswell G. Jacobs I. Milligan S. Parker M. Nat. Med. 2000; 6: 1368-1374Crossref PubMed Scopus (165) Google Scholar). Analysis of the nuclear receptor interaction domain of the p160 family led to the identification of three repeated motifs with a consensus sequence LXXLL in which L represents leucine andX represents any amino acid. The LXXLL motif has been found to be necessary and sufficient for ligand-dependent interactions with the nuclear receptor ligand binding domain (19Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1112) Google Scholar, 21Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1800) Google Scholar, 22Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1714) Google Scholar, 23Feng W. Ribeiro R.C.J. Wagner R.L. Nguyen H. Apriletti J.W. Fletterick R.J. Baxter J.D. Kushner P.J. West B.L. Science. 1998; 280: 1747-1749Crossref PubMed Scopus (520) Google Scholar, 24Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Genes Dev. 1998; 12: 3343-3356Crossref PubMed Scopus (834) Google Scholar, 25Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2304) Google Scholar). Structural studies of the PPARγ, ER, and T3R ligand binding domains complexed to fragments of the p160 nuclear receptor interaction domains revealed that these motifs form short α helices (22Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1714) Google Scholar, 23Feng W. Ribeiro R.C.J. Wagner R.L. Nguyen H. Apriletti J.W. Fletterick R.J. Baxter J.D. Kushner P.J. West B.L. Science. 1998; 280: 1747-1749Crossref PubMed Scopus (520) Google Scholar, 24Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Genes Dev. 1998; 12: 3343-3356Crossref PubMed Scopus (834) Google Scholar, 25Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2304) Google Scholar), with multiple LXXLL motifs within a single coactivator mediating cooperative interactions with nuclear receptor dimers or heterodimers. The LXXLL helix is oriented and positioned at each end by a “charge-clamp” consisting of a conserved lysine in helix 3 of the ligand binding domain and a conserved glutamate in the AF-2 helix. These residues grip the LXXLL helix so that the internal leucine residues can pack into a hydrophobic pocket in the receptor C terminus. Most nuclear receptor coactivators have proved to contain functionally important LXXLL helices, with additional residues contributing to binding specificity (e.g. Refs. 26McInerney E.M. Rose D.W. Flynn S.E. Westin S. Mullen T.-M. Krones A. Inostroza J. Torchia J. Nolte R.T. Assa-Munt N. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1998; 12: 3357-3368Crossref PubMed Scopus (531) Google Scholar and 27Heery D. Hoare S. J. Biol. Chem. 2001; 276: 6695-6702Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Furthermore, these contacts are sensitive to conformational changes induced by structurally distinct ligands. Many additional factors have been demonstrated to enhance nuclear receptor activity in functional assays, suggesting that they may serve as nuclear receptor coregulators (reviewed in Ref. 1Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Biochemical studies and protein-protein interaction screens suggest that many of these proteins function as components of large multiprotein complexes and that additional enzymatic activities may be important for their function. For example, the p160 protein GRIP1 can associate with arginine methyltransferase 1 (CARM1), which potentiates ligand-dependent transcription by several nuclear receptors (16Chen D. Ma H. Hong H. Koh S.S. Huang S.-M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2176Crossref PubMed Scopus (1019) Google Scholar). PRMTI, a second arginine methyltransferase to also functions as a nuclear receptor coactivator S. Chen D. Lee Y. Stallcup M. J. Biol. Chem. 2001; 276: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The CBP/p300 coactivators can recruit additional factors with such as the complexes V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar, R. Kalafus D. Ogliastro M.-H. Kioussi C. Xu L. Torchia J. Rosenfeld M.G. Glass C.K. Science. 1998; 279: 700-703Crossref PubMed Scopus (199) Google Scholar). The and of the recruited complexes may distinct acetyltransferases are required by transcription factors on specific gene targets E. Torchia J. Rose D.W. Xu L. Kurokawa R. E.M. Mullen T.M. Glass C.K. Rosenfeld M.G. Science. 1998; 279: PubMed Scopus Google Scholar). In to coactivator complexes that nucleosome remodeling or histone acetyltransferase other coactivator complexes have been identified. The characterized of these is the which the transcriptional activities of nuclear receptors and other transcription factors in J.D. M. S. Sci. S. A. 1999; PubMed Scopus Google Scholar, C. J. D. H. P. Genes Dev. 1998; 12: PubMed Scopus Google Scholar, P.A. S. S. W. R. Nature. 1999; PubMed Scopus Google Scholar). The complex is recruited to nuclear receptors in a ligand-dependent manner a referred to as which utilized LXXLL nuclear receptor interaction motifs J.W. F. Nature. PubMed Scopus Google Scholar, Y. C. S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of the gene in the results in at and initial studies in enhancer factors have suggested a defect in ligand-dependent thyroid hormone and receptor function P.A. S. S. W. R. Nature. 1999; PubMed Scopus Google Scholar, J.W. F. Nature. PubMed Scopus Google Scholar). other classes of transcription factors to activate transcription in these The complex consists of more than a a of which appears to that are components of other including and and have enzymatic functions J.D. M. S. Sci. S. A. 1999; PubMed Scopus Google Scholar, P.A. S. S. W. R. Nature. 1999; PubMed Scopus Google Scholar, M. S. W. J.D. S. Zhang Qin J. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). These factors may function to recruit RNA polymerase II to nuclear receptors. The complex is not associated with RNA polymerase II but can be in the presence of vitamin D receptor C. V. M. A. H. P. L. Nature. 1999; PubMed Scopus Google Scholar), suggesting a conformational or recruitment of additional components that interactions with RNA polymerase II complexes. As more than additional coactivators have been including proteins with activity and an RNA that appears to function as a coactivator (reviewed in Ref. 15McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1669) Google Scholar), is that protein complexes can act either or in in of the of of interactions J. W. D. R. G. Science. 2000; PubMed Scopus Google Scholar, Y. J. M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). potential for a of coactivators be for complexes to chromatin remodeling ligand-dependent recruitment of the so p160 factors, in with other factors such as p300, and p/CAF, required acetyltransferase recruitment of complexes such as the complex may function to enhance RNA polymerase II recruitment to the In addition, a number of factors have been that can act in a promoter-specific important enzymatic activities or protein-protein interactions and or with other complexes. For example, a coactivator both with nuclear receptors and CBP/p300 p160 factors or a a C-terminal domain and also contacts factors in the basal transcription complex S.L. J.E. L. J. D. B.H. Lee Lee J.W. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, F. P. M. E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). has revealed promoter-specific in or on specific CBP and p300 are functionally M. D. R.T. E. D.M. R. Cell. 1998; Full Text Full Text PDF Scopus Google Scholar, Y. A. T. T. Y. M. H. T. K. K. Mol. 1999; PubMed Scopus Google Scholar), and studies in suggest in retinoic acid receptor the biochemical studies that of transcription requires of p300 and CBP Y. Xu L. T. Torchia J. Kurokawa R. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). mice each of the p160 factors are are suggested in specific receptor functions J. Y. M.J. O'Malley B.W. Science. 1998; 279: PubMed Scopus Google Scholar, C. Y. J. N. V. S. T. Sci. S. A. 1999; PubMed Scopus Google Scholar, J. L. G. H. C. O'Malley B.W. Sci. S. A. 2000; PubMed Scopus Google Scholar, Rose D.W. F. T. W. D. A. Krones A. K. Rosenfeld R. Glass C.K. Rosenfeld M.G. Sci. S. A. 2000; PubMed Scopus Google for example, effects on cell events J. L. G. H. C. O'Malley B.W. Sci. S. A. 2000; PubMed Scopus Google Scholar, Rose D.W. F. T. W. D. A. Krones A. K. Rosenfeld R. Glass C.K. Rosenfeld M.G. Sci. S. A. 2000; PubMed Scopus Google Scholar). The for diverse coactivators in part, their and of coactivators, by the in CBP in specific cell H. 1999; PubMed Scopus Google Scholar). a is that the number of potential coregulators clearly exceeds the capacity for direct interaction by a single receptor. chromatin assays, complexes and complexes are found to be to estrogen receptor target genes in response to Y. J. M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). these complexes each estrogen receptor be by this in of the of of receptors J. W. D. R. G. Science. 2000; PubMed Scopus Google Scholar). A of on the glucocorticoid response elements of induced was by the of glucocorticoid receptors in a cell containing a of units J. W. D. R. G. Science. 2000; PubMed Scopus Google Scholar). might that is a exchange of receptors associated with which mediate a of and required for transcriptional activation. A of a promoter-specific coactivator has been provided by identification of the coactivator P. R. M. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, P. Zhang C. G. V. A. S. Cell. 1999; 98: Full Text Full Text PDF PubMed Scopus Google Scholar). is induced in by and as a with CBP and p160 factors, for and transcriptional activation. These the of specific of coactivators are required for regulated by the nuclear receptor. Several members of the nuclear receptor family appear to critical roles by actively gene as a ligand-independent on target genes or a ligand-dependent on other transcription units (Fig. 1). A for interacting proteins mediating these effects led to the cloning of the nuclear receptor corepressors and A. A. T. Torchia J. Kurokawa R. Kamei Y. A. M. Glass C.K. PubMed Scopus Google Scholar, J.D. Nature. PubMed Scopus Google Scholar, S. Mol. 1996; PubMed Scopus Google Scholar). These factors domains that can interact with mammalian homologues of proteins that have been in yeast to mediate transcriptional including and histone T. Mullen T.-M. M. C.D. Torchia J. Yang G. E. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: PubMed Scopus Google Scholar, L. D. R.J. Hassig C.A. Schreiber S.L. Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar). hormone can be with in the ligand binding domain of thyroid hormone receptor that enhance ligand-independent interactions with S.M. Mol. 1997; 11: PubMed Scopus Google Scholar). also repressive roles in the actions of other classes of transcription factors (reviewed in Ref. 1Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). has between and as deletion of the nuclear repression of specific genes K. T. A. V. R. Kurokawa R. V. F. E. S. G. Glass C.K. Rose D.W. Rosenfeld M.G. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of transcription in mice and the at specific in and that is a required of short repression by nuclear receptors and other factors. In addition, also appears to be required for a of repression suggest that specific of corepressor and histone mediate the gene-specific actions of on the of multiple and appear to be components of several distinct corepressor complexes. both proteins suggested to interact with complexes containing and specific on T. Mullen T.-M. M. C.D. Torchia J. Yang G. E. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: PubMed Scopus Google Scholar, L. D. R.J. Hassig C.A. Schreiber S.L. Cell. 1997; 89: Full Text Full Text PDF PubMed Scopus Google Scholar), complexes biochemical not contain or of in of three distinct complexes P. L. N. P. Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). complex and the second a histone and the complex activity. of complexes has also in the of at least three complexes C. M. S. Torchia J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Wang J. J. Qin J. J. J. 2000; 19: PubMed Scopus Google Scholar, M. W. W. E. M. Genes Dev. 2000; 14: PubMed Google Scholar, Y. V. L. Yang W. Glass C. Rosenfeld M. E. Sci. S. A. 2000; PubMed Scopus Google Scholar). complex and several other components found in the with the studies C. M. S. Torchia J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The second complex several additional components, including and a corepressor that has been to silencing T. Mullen T.-M. M. C.D. Torchia J. Yang G. E. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: PubMed Scopus Google Scholar). appears to be a of either or protein 1 a protein with and functional to the and corepressors M. W. W. E. M. Genes Dev. 2000; 14: PubMed Google Scholar). is also a of the complex M. W. W. E. M. Genes Dev. 2000; 14: PubMed Google Scholar, Y. V. L. Yang W. Glass C. Rosenfeld M. E. Sci. S. A. 2000; PubMed Scopus Google Scholar), as to the specific functions of in A specific conserved corepressor domain of and has also been to be capable of direct interaction with and Zhang J. M.G. Kouzarides T. Genes Dev. 2000; 14: Google Scholar, M. Nature. 1999; PubMed Scopus Google Scholar). In these suggest that with specific corepressor complexes are regulated and exhibit promoter and Two in the C-terminal regions of and appear to function to mediate interactions with thyroid hormone each containing a conserved consensus sequence that mediate interactions with thyroid and retinoic acid receptors M. Nature. 1999; PubMed Scopus Google Scholar, V. E.M. Kurokawa R. Krones A. Rose D.W. Lambert M.H. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1999; PubMed Scopus Google Scholar, L. H. J. C. E. J. V. K. R. J. Genes Dev. 1999; PubMed Scopus Google Scholar). This motif is to form an α helical than the LXXLL motif in nuclear receptor Biochemical that the motif in and and the LXXLL motif in coactivators for with of the corepressor helix to in the a that a second of corepressor can be recruited to such as estrogen receptor in the presence of J.D. M.R. D. D.M. Science. 1999; Scopus Google Scholar), also appears to be a required K. T. A. V. R. Kurokawa R. V. F. E. S. G. Glass C.K. Rose D.W. Rosenfeld M.G. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, K. Torchia J. Mullen T.-M. R. M. S. J. C.K. Glass C.K. Rosenfeld M.G. Rose D.W. Sci. S. A. 1998; 95: PubMed Scopus Google Scholar). of the estrogen receptor to or of the AF-2 helix (e.g. Ref. 25Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2304) Google Scholar), corepressor that coactivators and corepressors are themselves targets of multiple pathways, of which are illustrated in Fig. Regulation of coactivator and corepressor function a for of to specific of sequence-specific transcription factors. For example, the histone acetyltransferase activity of CBP has been suggested to be regulated by which coactivator activities the cell S. S. F. L. P. M. D. A. Nature. 1998; PubMed Scopus Google Scholar). The ability of CBP to serve as a coactivator of is enhanced in response to signaling a mechanism S. H. Science. 1998; PubMed Scopus Google Scholar). The p160 nuclear receptors can be in response to signaling of the to the Rose D.W. F. T. W. D. A. Krones A. K. Rosenfeld R. Glass C.K. Rosenfeld M.G. Sci. S. A. 2000; PubMed Scopus Google Scholar). of lysine residues to LXXLL motifs may the receptors. corepressors are targets of pathways, with activation of with a of a nuclear to a or S. H. Science. 1998; PubMed Scopus Google C. R.J. W. D. Cell. 1999; 98: Full Text Full Text PDF PubMed Scopus Google Scholar). The N terminus of has been to interact with the mammalian homologue of Drosophila in Y. R. H. P. M. D. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), in of Based on cotransfection can mediate a decrease of protein by a The of with nuclear receptors is by cell signaling events that can the and activity of activation of signaling that the of with estrogen receptors in the presence of the on the of the N terminus K. Torchia J. Mullen T.-M. R. M. S. J. C.K. Glass C.K. Rosenfeld M.G. Rose D.W. Sci. S. A. 1998; 95: PubMed Scopus Google Scholar). In addition, with or in of and of with 1 directly interactions between and nuclear receptors or S. M. Mol. Cell. Biol. 2000; 20: PubMed Scopus Google Scholar). Nuclear receptors can serve as or dependent the regulated exchange of binding of factors and characterized by distinct enzymatic and platform In to a ligand-dependent can interactions of specific coregulators with nuclear receptors or mediate their activity or between nuclear or The potential for exchange of nuclear receptors and has for the functional of multiple receptors of coregulatory complexes.

The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg(57) and Arg(59), two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization.