J
ournal Club

November 29, 2011

Farhoud Faraji

Kent Hunter
Gene Expression "Programs"
• Def: A gene network whose coordinated expression
affects the overall phenotype of a cell.
– P...
Objective
• To gain insight into the mechanisms of "epigenomic
control" of regulated programs of gene expression
• Model: ...
E2F coordinates transcription of genes
required for entry into S-phase
Regulation:

Also:
• Post-translational modificatio...
Suv39h1
• H3K9 methyltransferase
– Recruits Polycomb proteins -> Heterochromatin
spreads -> Genes silenced

• Known to ass...
Hypothesis
• Suv39h1 may function as a transcriptional
repressor of E2F1
• Support:
– Transgenic mouse phenotypes
– Comple...
Subnuclear Structures
• Polycomb group protein bodies (PcG bodies)
– Polycomb complex-associated heterochromatin
– Transcr...
PRC1 compacts chromatin and catalyses
the monoubiquitylation of histone H2A

Margueron & Reinberg. Nature. 2011
PRC1 compacts chromatin and catalyses
the monoubiquitylation of histone H2A

Pc2

Margueron & Reinberg. Nature. 2011
LncRNAs associate with polycomb group
proteins to modify chromatin state
ANRIL

HOTAIR

Aguilo et al. Cancer Res 2011
Gupt...
Known lncRNA functions

Wilusz et al. Genes Dev. 2009
Key Hypotheses
• Lysine methylation by histone methyltransferases can regulate the fate of nonhistone proteins
• Crosstalk...
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
Pc2 is a substrate for Suv39h1 in vitro
Which residue(s) of Pc2 are methylated?
Strategy:
1. Coexpress FLAG-Pc2 and wt Suv39h1 or
Suv39h1 H324L
1. anti-FLAG IP
2....
Suv39h1 dimethylates Pc2 at K191
Pc2 K191R mutant abolishes methylation
Suv39h1 specifically methylates Pc2 in vivo
Can histone demethylases
demethylate non-histone
substrates such as Pc2?
KDM4C demethylates Pc2K191me2 in vitro
Is Pc2K191me regulated under
conditions of E2F1 activation?
2
Pc2 methylation negatively correlates
with serum stimulation
Serum-induced demethylation of Pc2 on
growth-control gene promoters
Pc2K191me2

Pc2K191
Does KDM4C occupy growth control gene
promoters?
Does KDM4C demethylate Histone H3K9 in
response to serum stimulation?
No, KDM4C does
not demethylate
H3K9me2 on
growth-control
gene promoters in
response to serum
stimulation
KDM4C is recruited to growth control gene
promoters and demethylates Pc2K191me2
in response to serum stimulation.
But does...
KDM4C is required for serum-induced
growth-control gene expression
What about Pc2?
Pc2 is essential for serum-induced growth
control gene expression
Pc2 expression and KDM4C recruitment to
promoters are required for expression of
growth control genes.
But does the change...
Pc2 knockdown diminishes serum-induced
entry into S-phase of Hela cells ...
... and primary human fibroblasts
K191 methylation is a key regulator for cell
proliferation
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
Pc2K191me2 but not unmethylated Pc2
colocalizes with Bmi1 and Ring1A

Bmi1 and Ring1A are markers of
PcG bodies, a transcr...
Unmethylated Pc2 but not Pc2K191me2
colocalizes with SC35

SC35 is a marker of
ICGs, a transcriptionally active
chromatin ...
Do Pc2-associated E2F1 targets
relocate from PcG bodies to ICGs
upon serum stimulation?
Serum-induced relocation of growth-control
gene loci between PcG bodies and ICGs

Similar analysis
conducted with
PCNA and...
Pc2K191me2 but not unmethylated Pc2
interacts with components of PRC1
The effect of Pc2, Bmi1, and PHC1 KD on
relocation of MCM3 locus between PcG
bodies and ICGs

Suggests Pc2 is required for...
KDM4C is required for serum-induced
dissociation from PcG bodies
Demethylated Pc2 is required for
relocation of MCM3 locus
Recap
• Pc2 is essential for serum-induced growthcontrol gene expression and cell proliferation
• Suv39h1 dimethylates Pc2...
Recap (cont'd)
• KDM4C demethylates Pc2
• In the presence of serum, KDM4C is recruited
to promoters of growth-control gene...
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
Might Pc2 bind ncRNAs?
• Evidence suggests that ncRNA may contribute
to maintaining integrity of subnuclear bodies
• Chrom...
Pc2K191me2 and unmethylated Pc2 bind to
RNAs

TUG1 – previously
shown to repress
cell-cycle genes

NEAT2 – exclusive
signa...
Does Pc2 binding to ncRNAs affect its histone
code reading ability?

•
•

Histone array with TUG1 or NEAT2 alone showed no...
TUG1 colocalizes with PcG bodies
TUG1 KD:  growth-control gene expression and proliferation
(similar to Pc2 K191R mutation)
NEAT2 KD: growth-control gen...
Identifying TUG1 and NEAT2 interactors

Proteins involved in
transcriptional
repression

Proteins involved in
transcriptio...
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
Upshifted E2F1 band induced by serum stimulation is due
to post-translational modification
E2F1 SUMOylated in vitro and in vivo at K266
SUMOylation is required for E2F1 activation of
growth-control genes
Is E2F1 SUMOylation RNA-dependent?
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
Outline
1. The effects of Pc2 methylation/demethylation on
growth
2. The effects of Pc2 methylation/demethylation on its
s...
MS analysis of proteins exclusively copurified
with wt E2F1 but not K266R identifies CDCA7L
CDCA7L is recruited to growth-control gene
promoters and is required for growth control
gene activation in the presence of...
CDCA7L is required for serum-induced histone H2B
ubiquitination on growth-control gene promoters

CDCA7L links E2F1 SUMOyl...
Summary

Pc2 methylation/demethylation is a molecular
switch for the inactivation/activation of the
E2F1-mediated mitogeni...
Summary

In absence of mitogenic stimuli (i.e. serum)
• Non-histone methylation of Pc2 by Suv39h1
induces Pc2 to bind grow...
Summary (cont'd)
In the presence of mitogenic stimuli:
• KDM4C demethylates Pc2K191me2
• Unmethylated Pc2 associates with ...
Thank you for your attention

Questions?

Comments/criticisms to farajif@mail.nih.gov
11.29.11 - LCBG Department Journal Club
11.29.11 - LCBG Department Journal Club
11.29.11 - LCBG Department Journal Club
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  • Subnuclear structure-specific localization of TUG1 and NEAT2 may indicate that they serve as the actual docking sites responsible for relocation of Pc2-bound growth-control gene promoters.
  • 11.29.11 - LCBG Department Journal Club

    1. 1. J ournal Club November 29, 2011 Farhoud Faraji Kent Hunter
    2. 2. Gene Expression "Programs" • Def: A gene network whose coordinated expression affects the overall phenotype of a cell. – Proliferation, differentiation, cellular maturation • Regulated by a concerted interplay between transcription factors, epigenetic modulation, and signaling mediators
    3. 3. Objective • To gain insight into the mechanisms of "epigenomic control" of regulated programs of gene expression • Model: Serum-induced proliferation program
    4. 4. E2F coordinates transcription of genes required for entry into S-phase Regulation: Also: • Post-translational modification • Regulation of intracellular localization
    5. 5. Suv39h1 • H3K9 methyltransferase – Recruits Polycomb proteins -> Heterochromatin spreads -> Genes silenced • Known to associate with Rb/E2F complex • Suv39h1 transgenic mice – Overexpression:  homeostatic cell proliferation – h1/h2 dbl null:  viability at embryonic stage  growth as adults
    6. 6. Hypothesis • Suv39h1 may function as a transcriptional repressor of E2F1 • Support: – Transgenic mouse phenotypes – Complexes with Rb/E2F – Interacts with Cbx4(Pc2) of PRC1 • Cbx proteins may play a critical role in cell-cycle progression
    7. 7. Subnuclear Structures • Polycomb group protein bodies (PcG bodies) – Polycomb complex-associated heterochromatin – Transcriptionally repressive environment – Markers: Ring1A, Bmi1 (components of PRC1) • Interchromatin granules (ICGs) – Transcriptionally active environment – Thought to couple transcription and pre-mRNA splicing – Marker: SC35, NEAT2 Saitoh et al. Mol Bio Cell. 2004 Saurin et al. J Cell Biol. 1998
    8. 8. PRC1 compacts chromatin and catalyses the monoubiquitylation of histone H2A Margueron & Reinberg. Nature. 2011
    9. 9. PRC1 compacts chromatin and catalyses the monoubiquitylation of histone H2A Pc2 Margueron & Reinberg. Nature. 2011
    10. 10. LncRNAs associate with polycomb group proteins to modify chromatin state ANRIL HOTAIR Aguilo et al. Cancer Res 2011 Gupta et al Nature 2011
    11. 11. Known lncRNA functions Wilusz et al. Genes Dev. 2009
    12. 12. Key Hypotheses • Lysine methylation by histone methyltransferases can regulate the fate of nonhistone proteins • Crosstalk between subnuclear architectural features and ncRNA could be a possible mechanism for controlling mitogenic gene expression programs
    13. 13. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    14. 14. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    15. 15. Pc2 is a substrate for Suv39h1 in vitro
    16. 16. Which residue(s) of Pc2 are methylated? Strategy: 1. Coexpress FLAG-Pc2 and wt Suv39h1 or Suv39h1 H324L 1. anti-FLAG IP 2. Trypsin Digest 3. Mass-spectrometry
    17. 17. Suv39h1 dimethylates Pc2 at K191
    18. 18. Pc2 K191R mutant abolishes methylation
    19. 19. Suv39h1 specifically methylates Pc2 in vivo
    20. 20. Can histone demethylases demethylate non-histone substrates such as Pc2?
    21. 21. KDM4C demethylates Pc2K191me2 in vitro
    22. 22. Is Pc2K191me regulated under conditions of E2F1 activation? 2
    23. 23. Pc2 methylation negatively correlates with serum stimulation
    24. 24. Serum-induced demethylation of Pc2 on growth-control gene promoters Pc2K191me2 Pc2K191
    25. 25. Does KDM4C occupy growth control gene promoters?
    26. 26. Does KDM4C demethylate Histone H3K9 in response to serum stimulation?
    27. 27. No, KDM4C does not demethylate H3K9me2 on growth-control gene promoters in response to serum stimulation
    28. 28. KDM4C is recruited to growth control gene promoters and demethylates Pc2K191me2 in response to serum stimulation. But does recruitment of KDM4C affect the expression of these genes?
    29. 29. KDM4C is required for serum-induced growth-control gene expression
    30. 30. What about Pc2?
    31. 31. Pc2 is essential for serum-induced growth control gene expression
    32. 32. Pc2 expression and KDM4C recruitment to promoters are required for expression of growth control genes. But does the change in expression of these genes actually impact cell proliferation?
    33. 33. Pc2 knockdown diminishes serum-induced entry into S-phase of Hela cells ...
    34. 34. ... and primary human fibroblasts
    35. 35. K191 methylation is a key regulator for cell proliferation
    36. 36. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    37. 37. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    38. 38. Pc2K191me2 but not unmethylated Pc2 colocalizes with Bmi1 and Ring1A Bmi1 and Ring1A are markers of PcG bodies, a transcriptionally repressive chromatin environment
    39. 39. Unmethylated Pc2 but not Pc2K191me2 colocalizes with SC35 SC35 is a marker of ICGs, a transcriptionally active chromatin environment
    40. 40. Do Pc2-associated E2F1 targets relocate from PcG bodies to ICGs upon serum stimulation?
    41. 41. Serum-induced relocation of growth-control gene loci between PcG bodies and ICGs Similar analysis conducted with PCNA and MSH2
    42. 42. Pc2K191me2 but not unmethylated Pc2 interacts with components of PRC1
    43. 43. The effect of Pc2, Bmi1, and PHC1 KD on relocation of MCM3 locus between PcG bodies and ICGs Suggests Pc2 is required for relocation of MCM3 to ICGs
    44. 44. KDM4C is required for serum-induced dissociation from PcG bodies
    45. 45. Demethylated Pc2 is required for relocation of MCM3 locus
    46. 46. Recap • Pc2 is essential for serum-induced growthcontrol gene expression and cell proliferation • Suv39h1 dimethylates Pc2 at K191 • Methylation of Pc2K191 is a key regulator for the expression of E2F1-dependent growthcontrol genes and cell proliferation • In the absence of serum, Pc2K191me2 occupies growth control genes and relocates these transcription units to PcG body markers in a methyl-Pc2 dependent manner
    47. 47. Recap (cont'd) • KDM4C demethylates Pc2 • In the presence of serum, KDM4C is recruited to promoters of growth-control genes and is required for their expression • In the presence of serum, Pc2K191 occupies growth control genes and relocates these transcription units to ICGs in a KDM4C dependent manner
    48. 48. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    49. 49. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    50. 50. Might Pc2 bind ncRNAs? • Evidence suggests that ncRNA may contribute to maintaining integrity of subnuclear bodies • Chromodomains reported to bind RNA
    51. 51. Pc2K191me2 and unmethylated Pc2 bind to RNAs TUG1 – previously shown to repress cell-cycle genes NEAT2 – exclusive signature ncRNA for ICGs
    52. 52. Does Pc2 binding to ncRNAs affect its histone code reading ability? • • Histone array with TUG1 or NEAT2 alone showed no interaction with histone marks ncRNA guide chromodomain recognition of histone marks • switch preferential binding from repressive to activating histone marks
    53. 53. TUG1 colocalizes with PcG bodies
    54. 54. TUG1 KD:  growth-control gene expression and proliferation (similar to Pc2 K191R mutation) NEAT2 KD: growth-control gene expression and proliferation
    55. 55. Identifying TUG1 and NEAT2 interactors Proteins involved in transcriptional repression Proteins involved in transcriptional activation and splicing ncRNAs may impose a chromatin-remodeling environment by selectively interacting with chromatin modifier proteins
    56. 56. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    57. 57. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    58. 58. Upshifted E2F1 band induced by serum stimulation is due to post-translational modification
    59. 59. E2F1 SUMOylated in vitro and in vivo at K266
    60. 60. SUMOylation is required for E2F1 activation of growth-control genes
    61. 61. Is E2F1 SUMOylation RNA-dependent?
    62. 62. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    63. 63. Outline 1. The effects of Pc2 methylation/demethylation on growth 2. The effects of Pc2 methylation/demethylation on its subnuclear localization 3. The role of Pc2-ncRNA interaction in subnuclear localization 4. The role of Pc2 and ncRNA in E2F1-SUMOylation and gene activation 5. The effects of E2F1-SUMOylation on chromatin architecture
    64. 64. MS analysis of proteins exclusively copurified with wt E2F1 but not K266R identifies CDCA7L
    65. 65. CDCA7L is recruited to growth-control gene promoters and is required for growth control gene activation in the presence of serum
    66. 66. CDCA7L is required for serum-induced histone H2B ubiquitination on growth-control gene promoters CDCA7L links E2F1 SUMOylation and H2B ubiquitination in growth-control gene activation
    67. 67. Summary Pc2 methylation/demethylation is a molecular switch for the inactivation/activation of the E2F1-mediated mitogenic gene expression program.
    68. 68. Summary In absence of mitogenic stimuli (i.e. serum) • Non-histone methylation of Pc2 by Suv39h1 induces Pc2 to bind growth gene promoters • TUG1 sequesters Pc2K191me2 to PcG bodies thereby repressing transcriptional activation of growth control genes
    69. 69. Summary (cont'd) In the presence of mitogenic stimuli: • KDM4C demethylates Pc2K191me2 • Unmethylated Pc2 associates with NEAT2 and localizes to ICGs • Pc2 results in the Ubc9 and NEAT2-dependent SUMOylation of E2F1 • SUMO1-E2F1 binds CDCA7L which ubiquitinates H2B at promoters of growthcontrol genes • Transcription of growth control genes is activated and cells enter S-phase
    70. 70. Thank you for your attention Questions? Comments/criticisms to farajif@mail.nih.gov

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