The dynamics of early-state transcriptional changes and aggregate formation in a Huntington's disease cell model.

BACKGROUND: Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG expansion in the Huntingtin (HTT) gene. Proteolytic cleavage of mutant huntingtin (Htt) protein with an expanded polyglutamine (polyQ) stretch results in production of Htt fragments that aggregate and induce impaired ubiquitin proteasome, mitochondrial functioning and transcriptional dysregulation. To understand the time-resolved relationship between aggregate formation and transcriptional changes at early disease stages, we performed temporal transcriptome profiling and quantification of aggregate formation in living cells in an inducible HD cell model. RESULTS: Rat pheochromocytoma (PC12) cells containing a stably integrated, doxycycline-inducible, eGFP-tagged N-terminal human Htt fragment with an expanded polyQ domain were used to analyse gene expression changes at different stages of mutant Htt aggregation. At earliest time points after doxycycline induction no detectable aggregates and few changes in gene expression were observed. Aggregates started to appear at intermediate time points. Aggregate formation and subsequent enlargement of aggregates coincided with a rapid increase in the number of differentially expressed (DE) genes. The increase in number of large aggregates coincided with a decrease in the number of smaller aggregates whereas the transcription profile reverted towards the profile observed before mutant Htt induction. Cluster-based analysis of the 2,176 differentially expressed genes revealed fourteen distinct clusters responding differently over time. Functional enrichment analysis of the two major gene clusters revealed that genes in the up-regulated cluster were mainly involved in metabolic (antioxidant activity and cellular ketone metabolic processes) and genes in the down-regulated cluster in developmental processes, respectively. Promoter-based analysis of the identified gene clusters resulted in identification of a transcription factor network of which several previously have been linked to HD. CONCLUSIONS: We demonstrate a time-resolved relationship between Htt aggregation and changes in the transcriptional profile. We identified two major gene clusters showing involvement of (i) mitochondrial dysfunction and (ii) developmental processes implying cellular homeostasis defects. We identified novel and known HD-linked transcription factors and show their interaction with known and predicted regulatory proteins. Our data provide a novel resource for hypothesis building on the role of transcriptional key regulators in early stages of HD and possibly other polyQ-dependent diseases.

RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2alpha.

Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1alpha and HIF-2alpha are continuously synthesized in cells and degraded via the ubiquitin-proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels. HIF proteins are tightly regulated via post-translational modifications including phosphorylation, acetylation, prolyl-hydroxylation and ubiquitination. Here we show for the first time that exogenous and endogenous HIF-2alpha are also regulated via the ubiquitin-like modifier small ubiquitin-like modifiers (SUMO). Using mutational analysis, we found that K394, which is situated in the sumoylation consensus site LKEE, is the major SUMO acceptor site in HIF-2alpha. Functionally, sumoylation reduced the transcriptional activity of HIF-2alpha. Similar to HIF-1alpha, HIF-2alpha is regulated by the SUMO protease SENP1. The proteasome inhibitor MG132 strongly stabilized SUMO-2-conjugated HIF-2alpha during hypoxia but did not affect the total level of HIF-2alpha. The ubiquitin E3 ligases von Hippel-Lindau and RNF4 control the levels of sumoylated HIF-2alpha, indicating that sumoylated HIF-2alpha is degraded via SUMO-targeted ubiquitin ligases.

The ubiquitin-proteasome system is a key component of the SUMO-2/3 cycle.

Many proteins are regulated by a variety of post-translational modifications, and orchestration of these modifications is frequently required for full control of activity. Currently little is known about the combinatorial activity of different post-translational modifications. Here we show that extensive cross-talk exists between sumoylation and ubiquitination. We found that a subset of SUMO-2-conjugated proteins is subsequently ubiquitinated and degraded by the proteasome. In a screen for preferential SUMO-1 or SUMO-2 target proteins, we found that ubiquitin accumulated in purified SUMO-2 conjugates but not in SUMO-1 conjugates. Upon inhibition of the proteasome, the amount of ubiquitin in purified SUMO-2 conjugates increased. In addition, we found that endogenous SUMO-2/3 conjugates, but not endogenous SUMO-1 conjugates, accumulated in response to proteasome inhibitors. Quantitative proteomics experiments enabled the identification of 73 SUMO-2-conjugated proteins that accumulated in cells treated with proteasome inhibitors. Cross-talk between SUMO-2/3 and the ubiquitin-proteasome system controls many target proteins that regulate all aspects of nucleic acid metabolism. Surprisingly the relative abundance of 40 SUMO-2-conjugated proteins was reduced by proteasome inhibitors possibly because of a lack of recycled SUMO-2. We conclude that SUMO-2/3 conjugation and the ubiquitin-proteasome system are tightly integrated and act in a cooperative manner.

In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy.

The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.