1. J
ournal Club
November 29, 2011
Farhoud Faraji
Kent Hunter

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. Objective
• To gain insight into the mechanisms of "epigenomic
control" of regulated programs of gene expression
• Model: Serum-induced proliferation program

4. E2F coordinates transcription of genes
required for entry into S-phase
Regulation:
Also:
• Post-translational modification
• Regulation of intracellular localization

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

9. PRC1 compacts chromatin and catalyses
the monoubiquitylation of histone H2A
Pc2
Margueron & Reinberg. Nature. 2011

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. Known lncRNA functions
Wilusz et al. Genes Dev. 2009

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. 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. 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. Pc2 is a substrate for Suv39h1 in vitro

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. Suv39h1 dimethylates Pc2 at K191

18. Pc2 K191R mutant abolishes methylation

20. Suv39h1 specifically methylates Pc2 in vivo

21. Can histone demethylases
demethylate non-histone
substrates such as Pc2?

22. KDM4C demethylates Pc2K191me2 in vitro

23. Is Pc2K191me regulated under
conditions of E2F1 activation?
2

24. Pc2 methylation negatively correlates
with serum stimulation

25. Serum-induced demethylation of Pc2 on
growth-control gene promoters
Pc2K191me2
Pc2K191

26. Does KDM4C occupy growth control gene
promoters?

28. Does KDM4C demethylate Histone H3K9 in
response to serum stimulation?

29. No, KDM4C does
not demethylate
H3K9me2 on
growth-control
gene promoters in
response to serum
stimulation

30. 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?

31. KDM4C is required for serum-induced
growth-control gene expression

32. What about Pc2?

33. Pc2 is essential for serum-induced growth
control gene expression

34. 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?

35. Pc2 knockdown diminishes serum-induced
entry into S-phase of Hela cells ...

36. ... and primary human fibroblasts

37. K191 methylation is a key regulator for cell
proliferation

38. 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

39. 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

40. Pc2K191me2 but not unmethylated Pc2
colocalizes with Bmi1 and Ring1A
Bmi1 and Ring1A are markers of
PcG bodies, a transcriptionally
repressive chromatin environment

41. Unmethylated Pc2 but not Pc2K191me2
colocalizes with SC35
SC35 is a marker of
ICGs, a transcriptionally active
chromatin environment

42. Do Pc2-associated E2F1 targets
relocate from PcG bodies to ICGs
upon serum stimulation?

43. Serum-induced relocation of growth-control
gene loci between PcG bodies and ICGs
Similar analysis
conducted with
PCNA and MSH2

44. Pc2K191me2 but not unmethylated Pc2
interacts with components of PRC1

45. 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

46. KDM4C is required for serum-induced
dissociation from PcG bodies

47. Demethylated Pc2 is required for
relocation of MCM3 locus

48. 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

49. 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

50. 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

51. 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

52. Might Pc2 bind ncRNAs?
• Evidence suggests that ncRNA may contribute
to maintaining integrity of subnuclear bodies
• Chromodomains reported to bind RNA

53. Pc2K191me2 and unmethylated Pc2 bind to
RNAs
TUG1 – previously
shown to repress
cell-cycle genes
NEAT2 – exclusive
signature ncRNA for
ICGs

55. 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

56. TUG1 colocalizes with PcG bodies

57. TUG1 KD:  growth-control gene expression and proliferation
(similar to Pc2 K191R mutation)
NEAT2 KD: growth-control gene expression and proliferation

58. 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

59. 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

60. 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

61. Upshifted E2F1 band induced by serum stimulation is due
to post-translational modification

62. E2F1 SUMOylated in vitro and in vivo at K266

63. SUMOylation is required for E2F1 activation of
growth-control genes

64. Is E2F1 SUMOylation RNA-dependent?

65. 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

66. 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

67. MS analysis of proteins exclusively copurified
with wt E2F1 but not K266R identifies CDCA7L

68. CDCA7L is recruited to growth-control gene
promoters and is required for growth control
gene activation in the presence of serum

69. 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

70. Summary
Pc2 methylation/demethylation is a molecular
switch for the inactivation/activation of the
E2F1-mediated mitogenic gene expression
program.

71. 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

72. 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

73. Thank you for your attention
Questions?
Comments/criticisms to farajif@mail.nih.gov