1. Chromosomes and Gene Expression
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Asymmetry between the two acidic patches dictates the direction of nucleosome sliding by the ISWI chromatin remodeler

  1. Robert F Levendosky
  2. Gregory D Bowman  Is a corresponding author
  1. Johns Hopkins University, United States
Research Advance
Cite this article as: eLife 2019;8:e45472 doi: 10.7554/eLife.45472
5 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Design of nucleosomes with acidic patch mutations (APMs).

(A) The acidic patch of the nucleosome. In the overview, the surface is shown for one H2A/H2B dimer, with eight residues comprising the acidic patch highlighted in red. The acidic patch mutation (APM) consists of four alanine substitutions on H2A (E61A/E64A/D90A/E92A), highlighted in cyan. The structure shown is PDB code 1KX5 (Davey et al., 2002). (B) Strategy for making asymmetric nucleosomes using oriented hexasomes. As previously shown (Levendosky et al., 2016), hexasomes made with the Widom 601 (Lowary and Widom, 1998) preferentially lack an H2A/H2B dimer on the TA-poor side (here, the right side). Addition of either a wild type (gray) or APM H2A/H2B dimer (red) converts hexasomes to nucleosomes, allowing for production of all four combinations of H2A/H2B dimers with and without the APM. The locations of the H2A/H2B dimers and the H3/H4 tetramer on the Widom 601 is depicted in Figure 1—figure supplement 1.

https://doi.org/10.7554/eLife.45472.002
Figure 1—figure supplement 1
Arrangement of histone contacts with the Widom 601.

The approximate footprint from the histone core contacts of the H2A/H2B dimers (pink) and (H3/H4)2 tetramer (gray) are shown on the Widom 601 sequence (Lowary and Widom, 1998). In hexasomes, the sole H2A/H2B dimer preferentially resides on the TA-rich side of the Widom 601, shown here on the left.

https://doi.org/10.7554/eLife.45472.003
Mutations in the acidic patch slow down but do not disrupt centering ability of Chd1.

(A) Native gel nucleosome sliding assays using Chd1, with all four arrangements of APM and WT H2A/H2B dimers. Depending on which side the flanking DNA was on, the unique APM dimers were either on entry or exit sides, as indicated. The positions of the nucleosome cartoons indicate end-positioned nucleosomes in the gel, with the lower band representing residual hexasomes. Nucleosome centering is evidenced by slower migration in the gel. Reactions contained 40 nM hexasome, 60 nM H2A/H2B dimer, 200 nM Chd1, and 100 µM ATP. Note the different time series used (indicated above each gel), which helped capture sliding intermediates given the different reaction rates. Results are representative of three independent replicates. (B) Quantified data from (A) plotting the disappearance of end-positioned nucleosomes, overlaid with single exponential fits. The end-positioned band intensity was normalized to the total band intensity within each lane. Error bars show standard deviations from three replicates. (C) Mean observed rates ± standard deviations from fits obtained in (B). (D) Relative impact of APMs on Chd1 remodeling rates. Remodeling rates for WT/WT 0N80 and 80N0 were each scaled to 1. Rates for nucleosomes containing APM substitutions were scaled relative to WT/WT made with the same DNA construct. P-values *≤0.02; **<0.005; ***<0.0005.

https://doi.org/10.7554/eLife.45472.004
Figure 3 with 2 supplements
Asymmetry between the two nucleosome acidic patches disrupts nucleosome centering by ISWI remodelers.

(A) Native gel nucleosome sliding assays using SNF2h, with all four arrangements of WT and APM H2A/H2B dimers for 0N80 and 80N0 nucleosomes. Nucleosome centering corresponds to slower migration of nucleosome species. Asterisks highlight noticeable increases in free DNA. Remodeling reactions contained 40 nM hexasome, 80 nM H2A/H2B dimer, 1 µM SNF2h, and 2 mM ATP. Results are representative of two independent replicates. (B) Native gel nucleosome sliding experiments of 0N80 nucleosomes as in (A), but using Drosophila ACF. Remodeling reactions contained 40 nM hexasome, 70 nM H2A/H2B dimer, 100 nM ACF, and 2 mM ATP. Results are representative of two or more replicates. (C) ISWI remodelers but not Chd1 stimulate release of DNA from 0N80 nucleosomes with APM/WT histone cores. Shown is the accumulation of free DNA normalized to total band intensity from native gel experiments. All reactions tracking free DNA contained 40 nM hexasome, 70 nM H2A/H2B dimer, and 2 mM ATP with either 1 µM Snf2h, 100 nM ACF, or 200 nM Chd1. The mean and standard deviation of three or four replicates is plotted with single exponentials fits.

https://doi.org/10.7554/eLife.45472.005
Figure 3—figure supplement 1
For the ACF complex, association of the Acf1 subunit tracks with nucleosome sliding activity.

To ensure that the remodeling activity observed in experiments using ACF was not caused by the Iswi ATPase subunit independently, nucleosome sliding activity was monitored after retention of the FLAG-tagged Acf1 regulatory subunit with anti-FLAG beads. Native gel nucleosome sliding experiments (see Materials and methods section) were conducted using 10 nM ACF complex that was incubated for 30 min on spin columns either without anti-FLAG beads (A), with anti-FLAG beads (B), or with anti-FLAG beads that were blocked with 3xFLAG peptide (C). The loss of activity observed in (B) indicated that remodeling activity from ACF was due to the complete ACF complex. Experiments were carried out in duplicate.

https://doi.org/10.7554/eLife.45472.006
Figure 3—figure supplement 2
At sub-saturating concentrations, ACF shifts nucleosomes with asymmetric acidic patch mutations toward DNA ends.

Nucleosome sliding experiments monitored by native acrylamide gels were carried out with 10 nM ACF, 40 nM 0N80 APM hexasomes, 70 nM WT H2A/H2B dimer and 2 mM ATP. To see if the remodeling reaction had reached equilibrium, additional time points were taken after ~17 and 90 hr. These extended time points show a different distribution, suggesting that nucleosome bands begin moving back down the gel toward the end position. Although much slower than reactions performed with 100 nM ACF, these results are consistent with the finding that ACF initially centers and then shifts asymmetric APM/WT nucleosomes toward an end position. The rightmost lane contains nucleosomes from a reaction assembled without ATP that stayed at room temperature for 5400 min alongside the remodeling reaction. Shown is a representative of three independent replicates.

https://doi.org/10.7554/eLife.45472.007
Figure 4 with 3 supplements
SNF2h slides histone cores with asymmetric acidic patch mutations off DNA ends.

Histone mapping of SNF2h sliding reactions, performed with 0N80 nucleosomes having the four combinations of wild type and APM H2A/H2B dimers: (A) WT/WT, (B) WT/APM, (C) APM/WT, and (D) APM/APM. Shown are scans of urea denaturing gels, which report on the locations of the histone octamer based on sites of H2B(S53C) cross-linking. Two scans are shown for each nucleosome, based on Cy5 and FAM labels on the top and bottom DNA strands, respectively. Numbering beside the gels indicates the distances (bp) the histone octamer shifted relative to the starting position, with the midpoint of the 80 bp flanking DNA highlighted with a central orange bar. Each nucleosome was generated by addition of 200 nM wild type (gray) or APM (red) H2A/H2B dimers to 100 nM wild type or APM hexasome. Cartoon schematics below each gel indicate the composition and orientation of APM and wild type H2A/H2B dimers, the sites of H2B(S53C) cross-linking (black triangles), and interpretations of sliding reactions. Sites where the histone octamer moves off DNA ends are highlighted by dotted red boxes. Sliding reactions contained 1 µM SNF2h and 2 mM ATP, and reactions were quenched at 0, 4, and 64 min. These results are representative of two independent experiments. Analogous experiments using 80N0 nucleosomes are shown in Figure 4—figure supplement 1. Extended gels are shown in Figure 4—figure supplement 2 and Figure 4—figure supplement 3.

https://doi.org/10.7554/eLife.45472.008
Figure 4—figure supplement 1
SNF2h slides histone cores with single acidic patch mutations off DNA ends regardless of Widom 601 orientation.

Histone mapping of SNF2h sliding reactions, performed with 80N0 nucleosomes having the four combinations of wild type and APM H2A/H2B dimers: (A) WT/WT, (B) WT/APM, (C) APM/WT, and (D) APM/APM. These experiments are analogous to those of Figure 4 but with the opposite sequence orientation of the Widom 601 relative to flanking DNA. These experiments were carried out in duplicate alongside those in Figure 4. Extended gels are shown in Figure 4—figure supplement 2 and Figure 4—figure supplement 3.

https://doi.org/10.7554/eLife.45472.009
Figure 4—figure supplement 2
Extended gel images of histone mapping experiments using WT/WT and WT/APM nucleosomes.

These extended gels include the sequencing ladders used to determine the distances of shifted cross-links for experiments shown in Figure 4 and Figure 4—figure supplement 1. The dyad locations of unshifted nucleosomes are indicated.

https://doi.org/10.7554/eLife.45472.010
Figure 4—figure supplement 3
Extended gel images of histone mapping experiments using APM/WT and APM/APM nucleosomes.

These extended gels include the sequencing ladders used to determine the distances of shifted cross-links for experiments shown in Figure 4 and Figure 4—figure supplement 1. The dyad locations of unshifted nucleosomes are indicated.

https://doi.org/10.7554/eLife.45472.011
Figure 5 with 1 supplement
Schematics showing the spatial relationship between the remodeler ATPase motor, the two acidic patches, and flanking DNA of the nucleosome.

(A) A cartoon representation showing the two sides of a nucleosome with a remodeler ATPase motor bound at an SHL2 site. Broken arrows show paths connecting the ATPase motor with entry or exit DNA. (B) An illustration of how sliding the histone core off DNA ends impacts flanking DNA relative to the ATPase motor. As for ISWI, if action of the ATPase motor on one side of the nucleosome is severely limited due to an acidic patch mutation (brown "X"), DNA will be preferentially shifted in one direction due to ATPase action on the side with the wild type acidic patch (orange). Continued action on one side, with insufficient counteraction on the other side, will eventually pull all entry side DNA completely onto the histone core. If translocation continues without the need for flanking DNA on the entry side, the end of the DNA will eventually reach the ATPase motor at SHL2 (dotted outline).

https://doi.org/10.7554/eLife.45472.012
Figure 5—figure supplement 1
Chd1 remodelers have a longer linker connecting the ATPase motor and DNA-binding domain than ISWI.

Sequence alignments for a range of orthologs of ISWI (A) and Chd1 (B) chromatin remodelers, focused on the region between that ATPase motor and the DNA binding domain. Domain boundaries were taken from representative crystal structures: 3MWY (Hauk et al., 2010), 3TED (Sharma et al., 2011) and 1OFC (Grüne et al., 2003).

https://doi.org/10.7554/eLife.45472.013

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Antibodymouse monoclonal
ANTI-FLAG M2
Affinity Gel
Millipore SigmaCat #:2220(coupled to beads)
Recombinant DNA
reagent
core Widom 601
(uppercase) and
flanking DNA
sequences (lowercase)
Lowary and Widom, 19985’_gggatcctaatgaccaaggaaa
gcatgattcttcacaccgagttcatcc
cttatgtgatggaccctatacgcggc
cgcccTGGAGAATCCCGGTGCC
GAGGCCGCTCAATTGGTCGTA
GacAGCTCTAGCACCGCTTAAA
CGCACGTACGCGCTGTCCCCCG
CGTTTTAACCGCCAAGGGGAT
TACTCCCTAGTCTCCAGGCACG
TGTCAGATATATACATCCTGtgcat
gtattgaacagcgaccttgccggtgccag
tcggatagtgttccgagctcccactctaga
ggatccccgggtaccg_3’
Sequence-based
reagent
primer: Cy5-0-601IDT5’/5Cy5/TGGAGAATCCCGGTGCC
GAGGCCGCTCAAT
Sequence-based
reagent
primer: 601–80-FAMIDT5’/56-FAM/cggtacccggggatcctcta
gagtgggagc
Sequence-based
reagent
primer: 601–0-Cy5IDT5’/5Cy5/CAGGATGTATATATCTGAC
ACGTGCCTGGA
Sequence-based
reagent
primer: FAM-80–601IDT5’/56-FAM/gggatcctaatgaccaagg
aaagcatgatt
Peptide,
recombinant
protein
(Saccharomyces cerevisiae)
ScChd1118-1274McKnight et al., 2011from Saccharomyces cerevisiae
Peptide,
recombinant
protein
(Homo sapiens)
HsSNF2hYang et al., 2006
from Homo sapiens
Peptide,
recombinant
protein
(Drosophila melanogaster)
DmACFActif MotifCat #:31509from Drosophila melanogaster
Peptide,
recombinant
protein (Xenopus laevis)
XlHistone H2ALuger et al., 1997from Xenopus laevis
Peptide,
recombinant
protein (X. laevis)
XlHistone H2A-
E61A/E64A/D90A/
E92A ‘APM’
Girish et al., 2016
from Xenopus laevis
Peptide,
recombinant
protein (X. laevis)
XlHistone H2BLuger et al., 1997from Xenopus laevis
Peptide,
recombinant
protein (X. laevis)
XlHistone H2B-S53CKassabov et al., 2002
from Xenopus laevis
Peptide,
recombinant
protein (X. laevis)
XlHistone H3-C110ADechassa et al., 2010from Xenopus laevis
Peptide,
recombinant
protein (X. laevis)
XlHIistone H4Luger et al., 1997from Xenopus laevis
Commercial
assay or kit
Thermo sequenase
dye primer manual
cycle sequencing kit
AffymetrixCat #:79260
Chemical
compound, drug
NaClFisher ScientificCat #:S641-500
Chemical
compound, drug
Trizma baseSigmaCat #:T1503-1KG
Chemical
compound, drug
Boric AcidFisher ScientificCat #:A73-500
Chemical
compound, drug
MgCl2Fisher ScientificCat #:BP214-500
Chemical
compound, drug
EDTAThermo FisherCat #:AM9260G
Chemical
compound, drug
KClFisher ScientificCat #:P217-500
Chemical
compound, drug
DTTSigma AldrichCat #:D9779
Chemical
compound, drug
SucroseFisher ScientificCat #:S5-500
Chemical
compound, drug
Nonidet P-40Fisher ScientificCat #:MP1RIST1315
Chemical
compound, drug
BSANew England BiolabsCat #:B9000S
Chemical
compound, drug
Salmon Sperm
DNA
InvitrogenCat #:15632–011
Chemical
compound, drug
40% acrylamide/bis
solution 19:1
Bio-RadCat #:1610144
Chemical
compound, drug
UreaSigma AldrichCat #:U1250
Chemical
compound, drug
AcrylamideBio-RadCat #:1610101
Chemical
compound, drug
Bis N,N’-Methylene-Bis-
Acrylamide
Bio-RadCat #:1610201
Chemical
compound, drug
2-mercaptoethanolSigma AldrichCat #:M6250
Chemical
compound, drug
4-Azidophenacyl
bromide
Sigma AldrichCat #:A6057
Chemical
compound, drug
Phenol:Chloroform 5:1Sigma AldrichCat #:P1944
Chemical
compound, drug
Adenosine
triphosphate disodium
salt hydrate
Sigma AldrichCat #:A1852
Chemical
compound, drug
dNTPsInvitrogenCat #:10297–018
OtherMultipurpose Mini
Spin Columns
BioVisionCat # 6572–50
OtherHisPrep FF 16/10
(Nickel affinity)
GECat # 28-9365-51
OtherHisTrap HP, 5 ml
(Nickel affinity)
GECat # 17-5248-01
OtherHiTrap SP FF, 5 mlGECat # 17-5157-01
OtherHiTrap Q FF, 5 mlGECat # 17-5156-01
OtherHiLoad 16/600
Superdex 200,
prep grade
GECat # 28-9893-35
OtherHiLoad 16/600
Superdex 75,
prep grade
GECat # 28-9893-33
OtherHiPrep 26/10
Desalting
GECat # 17-5087-01
OtherHiPrep 16/10 Q FFGECat # 17-5190-01
OtherHiPrep 16/10 SP FFGECat # 17-5192-01
Software,
algorithm
ImageJimagej.nih.gov/ij/
Software,
algorithm
MathematicaWolfram

Data availability

All data generated in this study are included in the manuscript.

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