LeeAnn Boerma

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• LeeAnn Boerma
• Journal Club_FALL 2007
• Dr. Tim M. Townes

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• Small ubiquitin-related modifier protein (SUMO)
  is a protein modifier playing an important role
  in cellular functions such as cell cycle
  progression, nuclear-cytosolic transport, and
  transcriptional regulation
• Predominantly nuclear, SUMO targets include
  transcription factors, transcriptional
  co-regulators, chromosome-remodeling regulators
  and has historically been associated with protein
  stabilization
• Sumoylated lysines cannot be ubiquinated thus
  resulting in a stabilization of the target
  protein
• SUMO conjugation is a reversible phenomena-
  Sentrin/SUMO-specific proteases (SENPs)
• Much to be learned about the SUMO/deSUMO process

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• Yamaguchi et al 2005 show reduction of SENP1
  expression caused mice embryos to die between
  E12.5 and E14.5
• They note aberrant blood vessel development in
  the placenta
• This paper involves generation of SENP1 KO mice
  in an attempt to define the contribution of SENP1
  in development/mechanism elucidation

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SENP1 KO mice_gene-trap vector
Splice Acceptor Site
Polyadenylation site
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FIGURE 1. Severe Anemia in SENP-/- Embryos
SENP-/- live (dead) total offspring
SENP-/- embryos had 75 fewer erythrocytes than
SENP/- embryos
SENP1-/- embryos were paler/smaller than wt
death at days 13-15 of gestation
H and E Staining showing reduction in
erythropoietic foci and increase in apoptotic
cells
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FIGURE 2. SENP-/- Erythroid Progenitors Undergo
Apoptosis due to Epo Deficiency
Decreased ratio of CFU to BFU progenitors
Reduction in population of Ter-119 in SENP-/-
fetal livers
Anti Ter-119/TUNEL suggests SENP-/- more
apoptotic
Addition of Epo to media prevented reduction in
cell number
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• Does SENP1 directly regulates Epo expression in
  fetal liver?

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FIGURE 3. SENP1 Regulates transcription through
HIF1a
Reduction in Epo expression with
hypoxia/SiRNA_SENP1
Above suugests that Epo expression is regulated
by SENP1 through regulation of the HIF1a pathway
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• In normoxia, HIF1a is degraded in a proteosome
  dependent mechanism hypoxia was believed to
  stabilize HIF protein and increase its activity
  (1999-2004)
• Hypothesized that regulation of HIF1a to hypoxia
  was defective in SENP1-/- cells

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FIGURE 4. SENP1 is Essential for Ensuring HIF1a
Stability during Hypoxia
Hypoxia induced a greater increase in HIF1a
levels in wt cells
Hypoxia induced expression of HIF1a regulated
genes, VEGF and Glut-1, was reduced in SENP-/-
cells (below) HIF1a SUMOylation mutant restored
VEGF expression
Hypoxia induced HIF1a activity was significantly
reduced in SENP-/- cells
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• Concluded that SENP1 plays a critical role in
  regulating HIF1a during hypoxia
• Is SENP1 directly responsible for the decrease in
  HIF1a activity and stability in the SENP1-/-
  embryo?

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FIGURE 5. SUMOylated HIF1a Accumulates in SENP-/-
cells and Undergoes Proteasomal-dependent
Degradation under Hypoxia
Does SUMOylation lead to HIF1a degradation?
Hypoxia induced accumulation of SUMOylated HIFa
only seen in SENP-/- cells
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FIGURE 5. continued Does SUMOylation lead to
HIF1a degradation?
SUMOylated HIF1a accumulated in SENP-/- cells
after hypoxia and underwent proteosome dependent
degradation
Combined results suggest that SUMOylated HIF1a
was degraded in a proteosome dependent manner
during hypoxia, in the absence of deSUMOylation
factor, SENP1
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• Does SUMO and SENP1 regulate ubiquitination of
  HIF1a?

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FIGURE 6. VHL is required for Degradation of
SUMOylated HIF1a
HIF1a ubiquitination was regulated by SUMO-1 and
SENP1
SUMOylated HIF1a is degraded in a VHL-dependent
mechanism
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CONCLUSIONS

• SENP1 controls Epo production by regulation of
  HIF1a stability
• During normoxia, HIF1a is hydroxylated by PHD
  allowing its binding to VHL component of
  ubiquitin ligase complex- degradation
• In hypoxic conditions, HIF1a is summoned to the
  nucleus and SUMOylated, providing an alternative
  signal for degradation in a proteosome dependent
  manner
• SENP1 deSUMOylates HIF1a allowing it to escape
  degradation in low oxygen conditions and regulate
  Epo transcription

SUMOylation can target a protein for proteosome
degradation SUMOylation renders HIF1a
unstable, stabilized by deSUMOylation by SENP1