Resolving the Structure of Chromatin at the Centromere in Saccharomyces Cerevisiae Julian Haase Bloom Lab
Transcript
1. Resolving the Structure of Chromatin at the Centromere in
Saccharomyces Cerevisiae Julian Haase Bloom Lab
2. Cohesin enriched approximately 3-fold in a 50kb region
flanking the centromere (Weber et al., PLOS, 2006) How is
successful chromosome segregation achieved? Faithful segregation of
chromosomes to daughter cells is essential; failure leads to
aneuploidy, which can lead to cancer and diseases such as Downs
syndrome (trisomy 21) and Edwards syndrome (trisomy 18). The
centromere is a chromosomal locus that is required for mitosis and
acts as the site of kinetochore formation. The histone H3 variant
CENPA (Cse4) is incorporated here. The kinetochore is large
multi-protein complex consisting of over 70 proteins that are
recruited to the centromere. This then serves to mechanically link
the chromosomes to microtubules, through microtubule binding
components such as Ndc80. Once sister chromatids are properly
attached to opposite poles via microtubules, tension is generated
across the spindle. This tension fulfills checkpoints that allow
segregation to continue. Tension is achieved by holding sister
chromatids together prior to anaphase. The cohesin complex holds
sisters together.
3. Are centromere proximal lacO arrays bound together during
metaphase? How do we reconcile the 3-fold enrichment of cohesin at
centromeres with separated centromere proximal lacO arrays? Does
this model accurately portray live cell imaging of centromeres and
centromere proximal DNA? Outer Spots Spindle pole bodies Inner
spots CEN3 proximal lacO arrays (1.1kb from Cen3) Pearson et al.,
Journal Cell Biol., 2001 1 um
4. What is the path of DNA at the centromere? Can we visualize
cohesin enrichment at the centromere? How do we resolve the
organization of cohesin at the centromere? Deconvolution Model
Convolution What is the spatial confinement of pericentric
chromatin? What is the significance of kinetochore anisotropy? Are
there any mutants that regulate kinetochore anisotropy?
5. Chromosome Conformation Capture (3C) A method to detect the
interaction frequency between two points in the genome. This can be
used to infer the spatial arrangement and physical structure of a
chromatin fiber. 1) Crosslink 2) Digest 3) Ligate 4) Reverse
Crosslinks 5) PCR 1) Crosslink 2) Digest 3) Ligate 4) Reverse
Crosslinks 5) PCR Cen3 Cen3 15kb 23kb 50kb 50kb Decker et al.,
Science, 2002
6. What is the conformation of chromatin near centromeres? Yeh
and Haase et al., Current Biology, 2008 WT ChrIII 15kb (2.41) WT
ChrIII 23kb (1.64) WT ChrIII 50kb (0.25) mcd1-1 ChrIII 15kb (1.54)
nuf2-60 ChrIII 15kb (2.25) galCen3 ChrIII 15kb (1.18) WT ChrXI
12.3kb (2.49) Uncrosslinked (1.15) 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2
2.5 3 Pericentricvsarmexperimentalratio Pericentric vs arm control
ratio Chromosomal Interaction Frequency
7. Intra-strand cohesin Inter-strand cohesin C-loop Kinetochore
attachment Proposed path of centromeric DNA: the C-loop Accounts
for cohesin enrichment at pericentric DNA Predicts centromere
proximal lacO separation seen in live cells Predicts the increase
in chromosomal looping at pericentric DNA seen by 3C
8. What is the path of DNA at the centromere? Can we visualize
cohesin enrichment at the centromere? How do we resolve the
organization of cohesin at the centromere? Deconvolution Model
Convolution What is the spatial confinement of pericentric
chromatin? What is the significance of kinetochore anisotropy? Are
there any mutants that regulate kinetochore anisotropy?
9. What is the structure and function of the cohesin complex?
Intra-strand cohesin Inter-strand cohesin Four protein complex
Holds sister chromatids together Cleaved at anaphase onset
10. Can we detect cohesin enrichment at pericentric chromatin
by fluorescence? Yeh and Haase et al., Current Biology, 2008 End on
view Smc3 Spc29 Ndc80Smc3 Side on view Spc29Smc3
11. What is the path of DNA at the centromere? Can we visualize
cohesin enrichment at the centromere? How do we resolve the
organization of cohesin at the centromere? Deconvolution Model
Convolution What is the spatial confinement of pericentric
chromatin? What is the significance of kinetochore anisotropy? Are
there any mutants that regulate kinetochore anisotropy?
12. What are some limitations of light microscopy? Verdaasdonk
et al., Journal of Cellular Physiology, 2014 Airy discs and rings
The blurring of light by a microscope, the point spread function
(PSF), can be approximated by a Gaussian distribution Abbe
diffraction limit Spots within the Abbe limit appear as a single
diffraction limited spot.
13. How do we overcome the blurring of light? Verdaasdonk et
al., Journal of Cellular Physiology, 2014 Deconvolution restores
light blurred by the point spread function to the original point
source without loss of data. Smc3-GFP Original Deconvolved Smc3-GFP
before and after the application of nonlinear iterative
deconvolution
14. Can we get a clearer picture of the organization of cohesin
using deconvolution? Smc3 GFP side on view deconvolvedoriginal Smc3
GFP end on view deconvolvedoriginal
15. How is cohesin organized in the mitotic spindle? Smc3 Ndc80
Spc29 Smc3 Spc29 Smc3 By generating surface renders from
deconvolved images stepping through the spindle, we expect cohesin
is confined to a hollow barrel shaped region encompassing the
spindle.
16. What is the path of DNA at the centromere? Can we visualize
cohesin enrichment at the centromere? How do we resolve the
organization of cohesin at the centromere? Deconvolution Model
Convolution What is the spatial confinement of pericentric
chromatin? What is the significance of kinetochore anisotropy? Are
there any mutants that regulate kinetochore anisotropy?
17. Populate geometric shape with fluorophores Convolve with
experimental PSF Analyze and compare experimental and modelled
imagesExperimental PSF What is model convolution? Model convolution
provides subpixel accuracy of the position of fluorescently
labelled proteins. Stephens et al., MBoC, 2013 Takes the opposite
approach of deconvolution. It generates an understanding of the
possible fluorophore distributions that give rise to an
experimental image. This can be used to gain insight to the number
of molecules, the distribution of molecules, dynamics, and
more.
18. Can model convolution be used to predict the structure of
spindle components? Winey et al., 1995; Gardner et al., 2005
Stephens et al., MBoC, 2013
19. Stephens et al., MBoC, 2013 Can model convolution be used
to predict the structure of spindle components?
20. Stephens et al., MBoC, 2013 Can model convolution be used
to predict the structure of spindle components?
21. 550nm 500 nm What is the structural organization of cohesin
in the mitotic spindle? Cohesin is enriched 3-fold along
pericentric chromatin Imaging tells us cohesin is organized along
the spindle axis Using deconvolution, model convolution and surface
rendering we conclude cohesin is arrayed as a hollow cylinder
encompassing the spindle during metaphase. Yeh and Haase et al.,
Current Biology, 2008
22. What is the path of DNA at the centromere? Can we visualize
cohesin enrichment at the centromere? How do we resolve the
organization of cohesin at the centromere? Deconvolution Model
Convolution What is the spatial confinement of pericentric
chromatin? What is the significance of kinetochore anisotropy? Are
there any mutants that regulate kinetochore anisotropy?
23. Can we determine localization with sub pixel accuracy using
large population data sets? Using large population data sets
(n>200), we can generate positional density maps which show the
frequency with which something can be found at a given location.
Haase and Mishra et al., Current Biology, 2013
24. How do we validate heatmaps as a method we trust? Use
heatmaps to measure known values from in vivo measurements of
kinetochore components. Average discrepancy between heatmap and
SHREC values is 5.3 nm. Heatmaps faithfully reproduce measurements
from high localization accuracy techniques. Joglekar et al.,
Current Biology, 2009 Haase et al., Current Biology, 2012 SHREC -
Single molecule High Resolution Colocalization: two dimensional
(XY) measurement with high localization accuracy (10nm). 10 nm
Compare SHREC measurements of intra- kinetochore distances to
heatmap measurements to validate. Heatmap values SHREC values
25. How do chromatin heatmaps compare to cohesin localization?
LacO 1.1kb from Cen3 WT Distance (nm) Distance(nm) 0 130 259 389
518 648 778 907 648 518 389 259 130 0 -130 -259 -389 -518 -648 0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Y= 285.1nm 68.9nm X= 354.5nm
74.1nm n= 240 lacO 1.1kb from Cen3 LacO 1.8kb from Cen15 Metaphase
Distance (nm) Distance(nm) 0 130 259 389 518 648 778 907 648 518
389 259 130 0 -130 -259 -389 -518 -648 0 0.1 0.2 0.3 0.4 0.5 0.6
0.7 0.8 0.9 1 Y= 257.3nm 76.8nm X= 405.2nm 136.4nm n= 228 lacO
6.8kb from Cen15 LacO 3.8kb from Cen3 Metaphase Distance (nm)
Distance(nm) 0 130 259 389 518 648 778 907 648 518 389 259 130 0
-130 -259 -389 -518 -648 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Y=
326.2nm 110nm X= 420.6nm 175.3nm n= 208 lacO 8.8kb from Cen3
Centromere proximal chromatin fills a volumetric space similar to
that predicted by cohesin visualization -Width of cohesin barrel
encompasses the spread in the Y dimension of chromatin heatmaps
-Both cohesin and chromatin show decreased localization at the
spindle axis Stephens et al., JCB, 2011
26. What is the path of DNA at the centromere? Can we visualize
cohesin enrichment at the centromere? How do we resolve the
organization of cohesin at the centromere? Deconvolution Model
Convolution What is the spatial confinement of pericentric
chromatin? What is the significance of kinetochore anisotropy? Are
there any mutants that regulate kinetochore anisotropy?
27. Do inner and outer kinetochore components have the same
degree of anisotropy? Ndc80 Metaphase Ndc80 Anaphase C-Ndc80 575.56
N-Cse4 659.95 400 450 500 550 600 650 700 750 800 Spot Height in
Metaphase C-Ndc80 1.09 N-Cse4 1.23 1 1.1 1.2 1.3 Spot Anisotropy in
Anaphase (spot height/spot width) Broad Cse4 localization pattern
similar to that observed by Wisniewski, et al., eLife, 2014 Haase
et al., Current Biology, 2012 Cse4 Metaphase Cse4 Anaphase
28. Is kinetochore anisotropy the result of light blurring?
Broad non diffraction limited footprint of Cse4 remains distinct
after deconvolution when compared to Ndc80 Unlikely to be an
imaging artifact Metaphase Anaphase Ndc80Spc29 Ndc80
deconvolvedoriginal Ndc80Spc29 Ndc80 deconvolvedoriginal Ndc80Spc29
Ndc80 Ndc80Spc29 Ndc80 Cse4Spc29 Cse4 Cse4Spc29 Cse4 Cse4Spc29 Cse4
Cse4Spc29 Cse4
30. What is the path of DNA at the centromere? Can we visualize
cohesin enrichment at the centromere? How do we resolve the
organization of cohesin at the centromere? Deconvolution Model
Convolution What is the spatial confinement of pericentric
chromatin? What is the significance of kinetochore anisotropy? Are
there any mutants that regulate kinetochore anisotropy?
31. What makes Pat1 a candidate for anisotropy regulation at
the kinetochore? Wang et al., , 1996 Pilkington et al., , 2008
Mishra et al., Genetics, 2013 Structural component of the
kinetochore, and has a conserved region which mediates CEN
association Associates with centromeres in an NDC10 dependent
manner Loss of Pat1 delays sister chromatid separation, causes
errors in segregation, and leads to defects in structural integrity
of chromatin near the centromere. Protein Associated with
Topoisomerase II Involved in P-body assembly (non translating mRNAs
and decapping factors) More recently, Pat1 was found to have a role
in chromosome segregation independent of its function in P-body
assembly and translation repression
32. Does Pat1 play a role in kinetochore anisotropy? Ndc80
Metaphase Ndc80 pat1D Metaphase Ndc80 Anaphase Ndc80 pat1D Anaphase
C-Ndc80 1.09 C-Ndc80 pat1D 1.09 N-Cse4 1.23 N-Cse4 pat1D 1.06 1 1.1
1.2 1.3 Spot Anisotropy in Anaphase (spot height/spot width)
C-Ndc80 3,958.76 C-Ndc80 pat1D 3,321.30 N-Cse4 955.18 N-Cse4 pat1D
595.25 400 900 1400 1900 2400 2900 3400 3900 4400 Integrated Spot
Intensity C-Ndc80 575.56 C-Ndc80 pat1D 571.17 N-Cse4 659.95 N-Cse4
pat1D 569.87 400 450 500 550 600 650 700 750 800 Spot "Height" in
Metaphase Haase and Mishra et al., Current Biology, 2013 Absence of
Pat1 decreases Cse4 footprint to that of Ndc80 Cse4 Metaphase Cse4
pat1D Metaphase Cse4 Anaphase Cse4 pat1D Anaphase
37. Does Cse4 association to CEN change in the absence of Pat1?
Haase and Mishra et al., Current Biology, 2013 CEN association of
Cse4 is reduced in pat1D strains by ~60%. Centromeric levels of
Cse4 were assayed by ChIP analysis of Cse4-Myc at CEN1, 3 and 5 and
non-CEN DNA in wild type and pat1D strains.
38. Is depletion of Cse4 at the centromere in pat1D strains is
indicative of extra Cse4 molecules? 40% reduction in Cse4
fluorescence intensity upon loss of Pat1 Haase and Mishra et al.,
Current Biology, 2013 60% reduction of Cse4 at CEN by ChIP upon
loss of Pat1 Heatmaps show a change in Cse4 footprint to one
similar to that of Ndc80 upon loss of Pat1 Model convolution cannot
match WT Cse4 distribution without the addition of extra molecules
Pat1 regulates localization of an accessory pool of Cse4
39. 1x Kinetochore - Centromere Attachment Site 16x Kinetochore
Microtubules ~250nm diameter Interpolar Microtubules Pericentric
cohesin barrel surrounding spindle microtubules ~500nm diameter
Accessory molecules of Cse4 distributed along pericentric chromatin
Assembling the pieces Using a diverse set of techniques (3C,
deconvolution, model convolution, heatmaps) in conjunction with
widefield microscopy and ChIP, we reach the following conclusions:
C-loop
40. The Structure of Chromatin at the Centromere in
Saccharomyces Cerevisiae Julian Haase Bloom Lab University of North
Carolina at Chapel Hill
41. Acknowledgements Bloom Lab Current Members: Kerry Bloom
Elaine Yeh Josh Lawrimore Former Members: Ajit Joglekar Jolien
Verdaasdonk Andrew Stephens Rachel Haggerty UNC Computer Science
Department Russ Taylor Leandra Vicci Cory Quammen Basrai Lab Munira
Basrai Prashant Mishra UNC Physics Department Michael Falvo Salmon
Lab Ted Salmon Aussie Suzuki
42. Put extra slides after this point
43. Fluorescence light distribution in an image Point Spread
Function (PSF) of Light Light emitted from a point source is spread
out. 150nM 30nM Diffraction Limit Image resolution is limited by
the diffraction of light.
44. Chromatin conformation at Cen3 Cen3 15Kb Crosslinked Arm3
15Kb Crosslinked Cen3 15Kb uncrosslinked Arm3 15Kb uncrosslinked
15kb (n=10) 112249 52518 9485 7367 Crosslinked 15kb Cen3 vs. Arm3
Ratio 112249/52518 = 1.96 Uncrosslinked 15kb Cen3 vs. Arm3 Ratio
9485/7367 = 1.25 Crosslinked Ratio vs. Uncrosslinked Ratio
((1.96/1.25)-1)*100 = 56% From this, we can say that there is
increased physical proximity on either side of Cen3 relative to a
region on the arm. > Yeh and Haase et al., Current Biology,
2008
45. 3C product analysis Condition Cen3/Arm3 Crosslinked DNA
Cen3/Arm3 Uncrosslinked DNA Percent increase following crosslinking
xlinked-unxlinked Unxlinked Sample gel n P value WT 15kb 1.96 .18
1.25 .15 56.46% 10 4.46E-08 WT 23kb 1.50 .05 1.21 .03 23.81% 5
1.02E-05 WT 50kb 0.68 .25 1.21 .30 -43.81% 5 2.82E-04 aF 15kb 1.90
.21 1.22 .07 56.02% 10 1.33E-08 aF 23kb 1.52 .03 1.21 .03 25.91% 5
6.37E-07 aF 50kb 1.13 .05 1.21 .04 -6.58% 5 .002 ndc10-1 15kb 1.21
.08 1.18 .09 2.03% 10 .55 ndc10-1, aF 15kb 1.21 .03 1.20 .02 1.56%
10 .76 mcd1-1 15kb 1.44 .14 1.18 .05 22.17% 10 3.89E-05 gal cen
15kb 1.23 .05 1.21 .06 1.76% 10 .67 P xlinked A xlinked P unxlinked
A unxlinked Yeh and Haase et al., Current Biology, 2008
46. Degree of looping Yeh and Haase et al., Current Biology,
2008
47. 0 0.5 1 1.5 2 2.5 3 3.5 1 2 3 4 5 cen3vsarm3IntensityRatio
Actual Dilution of Cen3 vs Arm3 Intensity Ratios vs Actual
Dilutions Series1 3C artificial control
48. DIC Smc3 is organized around the spindle axis Confocal
images, 100nm steps Smc3 GFP Spc29 RFP Smc3GFP Spc29RFP Smc3
localizes as two lobes of fluorescence along either side of the
spindle axis, when the spindle is viewed side on. The lobes are
inside the spindle pole bodies, indicating the cohesin structure is
shorter than the spindle. Find more/better images to show here Yeh
and Haase et al., Current Biology, 2008
49. DIC This end-on view suggests cohesin is organized in a
cylindrical array. Confocal images, 100nm steps Smc3 localizes as a
hollow circle when viewed end on. Spindle pole bodies can be seen
directly in the center of this structure. This doughnut shape, when
considered along with the bi-lobed distribution, suggests cohesin
forms a cylinder that wraps around the spindle. Smc3 GFP Spc29 RFP
Find more/better images to show here. Smc3GFP Spc29RFP Yeh and
Haase et al., Current Biology, 2008
50. Images of cohesin + kinetochores Yeh and Haase et al.,
Current Biology, 2008 11.10.11 #12611.10.11 #44
51. WT cohesin time series Frap Scope, unbinned t0m t3m t6m t9m
t12m t15m t18m t0m t5m t10m t15m t20m
52. Side On End On Width (nm) 417 485 St Dev (nm) 36 76 Side On
End On Original Deconvolved Original Deconvolved Width (nm) 417
538* 485 559* St Dev (nm) 36 60 76 30
53. Confocal WT End On view Smc3 GFP Smc3 GFP deconvolved Smc3
GFP Smc3 GFP deconvolved
54. Confocal WT Side On view Smc3 GFP Smc3 GFP deconvolved Smc3
GFP Smc3 GFP deconvolved
56. End On Decon Width (inclusive) pixels nm 7.75 502 10 648
8.5 551 8 518 8 518 9.5 616 Average Average 8.63 559 Side On Decon
Width (inclusive) pixels nm 9 583 8 518 8 518 8.5 551 8 518 9 583 8
518 8 518 Average Average 8.31 538
57. Microscopy Assisted by Graphics and Interactive Convolution
(MAGIC) How can we test if this proposed structure generates the
fluorescent pattern we see in vivo? With MAGIC! Model Fluorescent
Image Special thanks to Cory Quammen and Russ Taylor, members of
the Nanoscale Science Research Group, part of the Computer Science
Department at UNC-Chapel Hill Magic Image
58. Simulations of clustering Andrew Stephens Cory W. Quammen
& Russell M. Taylor II UNC Computer Science
59. Model convolution of mitotic spindle structures Stephens et
al., MBoC, 2013
60. Wildtype spot shapes, perpendicular to spindle axis
Metaphase: Cse4 GFP Width: 655nm Max intensity: 352 Integrated
Intensity: 16741 Anaphase: Cse4 GFP Width: 642nm (1.98% decrease
from metaphase) Max Intensity: 387 (9.73% increase from metaphase)
Integrated Intensity:17496 (4.51% increase from metaphase)
Metaphase: Ndc80 GFP Width: 576nm Max intensity: 748 Integrated
Intensity: 35753 Anaphase: Ndc80 GFP Width: 556nm (3.47% decrease
from metaphase) Max Intensity: 820 (9.59% increase from metaphase)
Integrated Intensity:37299 (4.32% increase from metaphase) Decrease
in width represents decrease in radius of spherical structure, not
just a 2D decrease!
61. Metaphase: WT Cse4 GFP Width: 655nm Max intensity: 352
Integrated Intensity: 16741 Metaphase: mre11D Cse4 GFP Width: 541nm
Max intensity: Integrated Intensity: Wildtype vs mre11D spot
shapes, perpendicular to spindle axis Anaphase: Cse4 GFP Width:
642nm (1.98% decrease from metaphase) Max Intensity: 387 (9.73%
increase from metaphase) Integrated Intensity:17496 (4.51% increase
from metaphase) Anaphase: mre11D Cse4 GFP Width: 537nm Max
intensity: Integrated Intensity:
66. Heatmap Validation Haase and Mishra et al., Current
Biology, 2013
67. Haase and Mishra et al., Current Biology, 2013 Heatmap
Validation
68. Cse4 transcription is not affected in pat1D strains
Transcription of the CSE4 gene is not affected in pat1 strains.
Total RNA was extracted from wild type and pat1D strains as
determined by qRT- PCR. Haase and Mishra et al., Current Biology,
2013
69. Future Directions By what mechanisms are accessory
molecules of Cse4 regulated? -Pat1 prevents ubiquitination of Cse4?
-examine rates of ubiquitination in WT vs pat1D -does increasing
rate of ubiquitination in WT cells replicate Cse4 distribution in
pat1D? Do accessory Cse4 molecules serves as a rapid response to
detachment events mre11? Super Resolution Imaging Structured
Illumination Microscopy (SIM)