Multiple mechanisms of growth hormone-regulated gene transcription

Diverse physiological actions of growth hormone (GH) are mediated by changes in gene transcription. Transcription can be regulated at several levels, including post-translational modification of transcription factors, and formation of multiprotein complexes involving transcription factors, co-regulators and additional nuclear proteins; these serve as targets for regulation by hormones and signaling pathways. Evidence that GH regulates transcription at multiple levels is exemplified by analysis of the proto-oncogene c-fos. Among the GH-regulated transcription factors on c-fos, C/EBPbeta appears to be key, since depletion of C/EBPbeta by RNA interference blocks the stimulation of c-fos by GH. The phosphorylation state of C/EBPbeta and its ability to activate transcription are regulated by GH through MAPK and PI3K/Akt-mediated signaling cascades. The acetylation of C/EBPbeta also contributes to its ability to activate c-fos transcription. These and other post-translational modifications of C/EBPbeta appear to be integrated for regulation of transcription by GH. The formation of nuclear proteins into complexes associated with DNA-bound transcription factors is also regulated by GH. Both C/EBPbeta and the co-activator p300 are recruited to c-fos in response to GH, altering c-fos promoter activation. In addition, GH rapidly induces spatio-temporal re-localization of C/EBPbeta within the nucleus. Thus, GH-regulated gene transcription mediated by C/EBPbeta reflects the integration of diverse mechanisms including post-translational modifications, modulation of protein complexes associated with DNA and re-localization of gene regulatory proteins. Similar integration involving other transcription factors, including Stats, appears to be a feature of regulation by GH of other gene targets.Fil: Ceseña, Teresa I.. University of Michigan; Estados UnidosFil: Cui, Tracy Xiao. University of Michigan; Estados UnidosFil: Piwien Pilipuk, Graciela. Fundación Instituto Leloir; ArgentinaFil: Kaplani, Julianne. University of Michigan; Estados UnidosFil: Calinescu, Anda Alexandra. Michigan State University; Estados UnidosFil: Huo, Jeffrey S.. University of Michigan; Estados UnidosFil: Iñiguez Lluhí, Jorge A.. University of Michigan; Estados UnidosFil: Kwok, Roland. University of Michigan; Estados UnidosFil: Schwartz, Jessica. University of Michigan; Estados Unido

SUMOylation of the mitochondrial fission protein Drpl occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle

Dynamin‐related protein (Drp) 1 is a key regulator of mitochondrial fission and is composed of GTP‐binding, Middle, insert B, and C‐terminal GTPase effector (GED) domains. Drpl associates with mitochondrial fission sites and promotes membrane constriction through its intrinsic GTPase activity. The mechanisms that regulate Drpl activity remain poorly understood but are likely to involve reversible post‐translational modifications, such as conjugation of small ubiquitin‐like modifier (SUMO) proteins. Through a detailed analysis, we find that Drpl interacts with the SUMO‐conjugating enzyme Ubc9 via multiple regions and demonstrate that Drpl is a direct target of SUMO modification by all three SUMO isoforms. While Drpl does not harbor consensus SUMOylation sequences, our analysis identified2 clusters of lysine residues within the B domain that serve as noncanonical conjugation sites. Although initial analysis indicates that mitochondrial recruitment of ectopically expressed Drpl in response to staurosporine is unaffected by loss of SUMOylation, we find that Drpl SUMOylation is enhanced in the context of the K38A mutation. This dominant‐negative mutant, which is deficient in GTP binding and hydrolysis, does not associate with mitochondria and prevents normal mitochondrial fission. This finding suggests that SUMOylation of Drpl is linked to its activity cycle and is influenced by Drpl localization.—Figueroa‐Romero, C., Iniguez‐Lluhi, J. A., Stadler, J., Chang, C.‐R., Arnoult, D., Keller, P. J., Hong, Y., Blackstone, C., Feldman, E. L. SUMOylation of the mitochondrial fission protein Drpl occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle. FASEB J. 23, 3917–3927 (2009). www.fasebj.orgPeer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154272/1/fsb2fj09136630.pd

Disrupting SUMOylation enhances transcriptional function and ameliorates polyglutamine androgen receptor-mediated disease.

Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that are sites of SUMOylation. We determined that replacement of these residues by arginine enhances polyQ AR activity as a hormone-dependent transcriptional regulator. In a murine model, disruption of polyQ AR SUMOylation rescued exercise endurance and type I muscle fiber atrophy; it also prolonged survival. These changes occurred without overt alterations in polyQ AR expression or aggregation, revealing the favorable trophic support exerted by the ligand-activated receptor. Our findings demonstrate beneficial effects of enhancing the transcriptional function of the ligand-activated polyQ AR and indicate that the SUMOylation pathway may be a potential target for therapeutic intervention in SBMA

A Small Conserved Surface in SUMO Is the Critical Structural Determinant of Its Transcriptional Inhibitory Properties

Small ubiquitin-like modifier (SUMO) modification of sequence-specific transcription factors has profound regulatory consequences. By providing an intrinsic inhibitory function, SUMO isoforms can suppress transcriptional activation, particularly at promoters harboring multiple response elements. Through a comprehensive structure-function analysis, we have identified a single critical sector along the second beta sheet and the following alpha helix of SUMO2. This distinct surface is defined by four basic residues (K33, K35, K42, R50) that surround a shallow pocket lined by aliphatic (V30, I34) and polar (T38) residues. Substitutions within this area specifically and dramatically affected the ability of both SUMO2 and SUMO1 to inhibit transcription and revealed that the positively charged nature of the key basic residues is the main feature responsible for their functional role. This highly conserved surface accounts for the inhibitory properties of SUMO on multiple transcription factors and promoter contexts and likely defines the interaction surface for the corepressors that mediate the inhibitory properties of SUMO

Three Amino Acid Substitutions Selectively Disrupt the

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Bifunctional Ligands Allow Deliberate Extrinsic Reprogramming of the Glucocorticoid Receptor

Therapies based on conventional nuclear receptor ligands are extremely powerful, yet their broad and long-term use is often hindered by undesired side effects that are often part of the receptor's biological function. Selective control of nuclear receptors such as the glucocorticoid receptor (GR) using conventional ligands has proven particularly challenging. Because they act solely in an allosteric manner, conventional ligands are constrained to act via cofactors that can intrinsically partner with the receptor. Furthermore, effective means to rationally encode a bias for specific coregulators are generally lacking. Using the (GR) as a framework, we demonstrate here a versatile approach, based on bifunctional ligands, that extends the regulatory repertoire of GR in a deliberate and controlled manner. By linking the macrolide FK506 to a conventional agonist (dexamethasone) or antagonist (RU-486), we demonstrate that it is possible to bridge the intact receptor to either positively or negatively acting coregulatory proteins bearing an FK506 binding protein domain. Using this strategy, we show that extrinsic recruitment of a strong activation function can enhance the efficacy of the full agonist dexamethasone and reverse the antagonist character of RU-486 at an endogenous locus. Notably, the extrinsic recruitment of histone deacetylase-1 reduces the ability of GR to activate transcription from a canonical GR response element while preserving ligand-mediated repression of nuclear factor-κB. By providing novel ways for the receptor to engage specific coregulators, this unique ligand design approach has the potential to yield both novel tools for GR study and more selective therapeutics