Jacob Seeler

The Nucleoporin RanBP2 Has SUMO1 E3 Ligase Activity

Posttranslational modification with SUMO1 regulates protein/protein interactions, localization, and stability. SUMOylation requires the E1 enzyme Aos1/Uba2 and the E2 enzyme Ubc9. A family of E3-like factors, PIAS proteins, was discovered recently. Here we show that the nucleoporin RanBP2/Nup358 also has SUMO1 E3-like activity. RanBP2 directly interacts with the E2 enzyme Ubc9 and strongly enhances SUMO1-transfer from Ubc9 to the SUMO1 target Sp100. The E3-like activity is contained within a 33 kDa domain of RanBP2 that lacks RING finger motifs and does not resemble PIAS family proteins. Our findings place SUMOylation at the cytoplasmic filaments of the NPC and suggest that, at least for some substrates, modification and nuclear import are linked events.

Human Male Infertility Associated with Mutations in NR5A1 Encoding Steroidogenic Factor 1

One in seven couples worldwide are infertile, and male factor infertility accounts for approximately 30%-50% of these cases. Although many genes are known to be essential for gametogenesis, there are surprisingly few monogenic mutations that have been conclusively demonstrated to cause human spermatogenic failure. A nuclear receptor, NR5A1 (also called steroidogenic factor 1), is a key transcriptional regulator of genes involved in the hypothalamic-pituitary-steroidogenic axis, and it is expressed in the steroidogenic tissue of the developing and adult human gonad. Mutations of NR5A1 have been reported in 46,XY disorders of sex development and in 46,XX primary ovarian insufficiency. To test the hypothesis that mutations in NR5A1 cause male infertility, we sequenced NR5A1 in 315 men with idiopathic spermatogenic failure. We identified seven men with severe spermatogenic failure who carried missense mutations in NR5A1. Functional studies indicated that these mutations impaired NR5A1 transactivational activity. We did not observe these mutations in more than 4000 control alleles, including the entire coding sequence of 359 normospermic men and 370 fertile male controls. NR5A1 mutations are found in approximately 4% of men with otherwise unexplained severe spermatogenic failure.

An Acetylation/Deacetylation-SUMOylation switch through a phylogenetically conserved ΨKxEP motif in the tumor suppressor HIC1 (Hypermethylated in Cancer 1) regulates transcriptional repression activity

ABSTRACT Tumor suppressor HIC1 (hypermethylated in cancer 1) is a gene that is essential for mammalian development, epigenetically silenced in many human tumors, and involved in a complex pathway regulating P53 tumor suppression activity. HIC1 encodes a sequence-specific transcriptional repressor containing five Krüppel-like C2H2 zinc fingers and an N-terminal BTB/POZ repression domain. Here, we show that endogenous HIC1 is SUMOylated in vivo on a phylogenetically conserved lysine, K314, located in the central region which is a second repression domain. K314R mutation does not influence HIC1 subnuclear localization but significantly reduces its transcriptional repression potential, as does the mutation of the other conserved residue in the ψKXE consensus, E316A, or the overexpression of the deSUMOylase SSP3/SENP2. Furthermore, HIC1 is acetylated in vitro by P300/CBP. Strikingly, the K314R mutant is less acetylated than wild-type HIC1, suggesting that this lysine is a target for both SUMOylation and acetylation. We further show that HIC1 transcriptional repression activity is positively controlled by two types of deacetylases, SIRT1 and HDAC4, which increase the deacetylation and SUMOylation, respectively, of K314. Knockdown of endogenous SIRT1 by the transfection of short interfering RNA causes a significant loss of HIC1 SUMOylation. Thus, this dual-deacetylase complex induces either a phosphorylation-dependent acetylation-SUMOylation switch through a ψKXEXXSP motif, as previously shown for MEF2, or a phosphorylation-independent switch through a ψKXEP motif, as shown here for HIC1, since P317A mutation severely impairs HIC1 acetylation. Finally, our results demonstrate that HIC1 is a target of the class III deacetylase SIRT1 and identify a new posttranslational modification step in the P53-HIC1-SIRT1 regulatory loop.

PARP-1 transcriptional activity is regulated by sumoylation upon heat shock

Heat shock and other environmental stresses rapidly induce transcriptional responses subject to regulation by a variety of post‐translational modifications. Among these, poly(ADP‐ribosyl)ation and sumoylation have received growing attention. Here we show that the SUMO E3 ligase PIASy interacts with the poly(ADP‐ribose) polymerase PARP‐1, and that PIASy mediates heat shock‐induced poly‐sumoylation of PARP‐1. Furthermore, PIASy, and hence sumoylation, appears indispensable for full activation of the inducible HSP70.1 gene. Chromatin immunoprecipitation experiments show that PIASy, SUMO and the SUMO‐conjugating enzyme Ubc9 are rapidly recruited to the HSP70.1 promoter upon heat shock, and that they are subsequently released with kinetics similar to PARP‐1. Finally, we provide evidence that the SUMO‐targeted ubiquitin ligase RNF4 mediates heat‐shock‐inducible ubiquitination of PARP‐1, regulates the stability of PARP‐1, and, like PIASy, is a positive regulator of HSP70.1 gene activity. These results, thus, point to a novel mechanism for regulating PARP‐1 transcription function, and suggest crosstalk between sumoylation and RNF4‐mediated ubiquitination in regulating gene expression in response to heat shock.

SUMO, the Three Rs and Cancer

SUMO modification (sumoylation) plays important roles in nucleo-cytoplasmic transport, maintenance of sub-nuclear architecture, the regulation of gene expression and in DNA replication, repair and recombination. Here we review recent evidence for SUMOs role in protecting genomic integrity at both the chromosomal and the DNA level. Furthermore, the involvement of sumoylation and of specific SUMO targets in cancer is discussed.

Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation

Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localization of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show an increase in both cell size and global protein levels. Strikingly, we found that in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.

Arkadia, a Novel SUMO-Targeted Ubiquitin Ligase Involved in PML Degradation

ABSTRACT Arkadia is a RING domain E3 ubiquitin ligase that activates the transforming growth factor β (TGF-β) pathway by inducing degradation of the inhibitor SnoN/Ski. Here we show that Arkadia contains three successive SUMO-interacting motifs (SIMs) that mediate noncovalent interaction with poly-SUMO2. We identify the third SIM (VVDL) of Arkadia to be the most relevant one in this interaction. Furthermore, we provide evidence that Arkadia can function as a SUMO-targeted ubiquitin ligase (STUBL) by ubiquitinating SUMO chains. While the SIMs of Arkadia are not essential for SnoN/Ski degradation in response to TGF-β, we show that they are necessary for the interaction of Arkadia with polysumoylated PML in response to arsenic and its concomitant accumulation into PML nuclear bodies. Moreover, Arkadia depletion leads to accumulation of polysumoylated PML in response to arsenic, highlighting a requirement of Arkadia for arsenic-induced degradation of polysumoylated PML. Interestingly, Arkadia homodimerizes but does not heterodimerize with RNF4, the other STUBL involved in PML degradation, suggesting that these two E3 ligases do not act synergistically but most probably act independently during this process. Altogether, these results identify Arkadia to be a novel STUBL that can trigger degradation of signal-induced polysumoylated proteins.

Correction for Erker et al., “Arkadia, a Novel SUMO-Targeted Ubiquitin Ligase Involved in PML Degradation”

Volume 33, no. 11, p. 2163–2177, 2013, https://doi.org/10.1128/MCB.01019-12. Page 2168, Fig. 2B: The GFP control in the left panel was inadvertently used to illustrate the GFP control in the right panel during figure construction. The correct GFP control for the right panel is shown below. Citation Erker Y, Neyret-Kahn H, Seeler JS, Dejean A, Atfi A, Levy L. 2017. Correction for Erker et al., “Arkadia, a novel SUMO-targeted ubiquitin ligase involved in PML degradation.” Mol Cell Biol 37:e00193-17. https://doi.org/10 .1128/MCB.00193-17. Copyright