Additional file 2 of The chromatin remodeler Ino80 mediates RNAPII pausing site determination

Additional file 2. Review history

Additional file 1 of The chromatin remodeler Ino80 mediates RNAPII pausing site determination

Additional file 1 : Fig. S1. PRO-seq analysis in S. cerevisiae upon the loss of Spt4p. Fig. S2. PRO-seq analysis in S. pombe. Fig. S3. PRO-seq analysis in mESCs. Fig. S4. PRO-cap detects the precise transcription initiation sites genome-wide. Fig. S5. PRO-seq is highly correlated with Rpb3p NET-seq and ChIP-exo in S. cerevisiae. Fig. S6. Correlation of promoter-proximal PRO-seq pattern with nucleosome architecture and gene activity. Fig. S7. AID system is employed to investigate the immediate effect upon Ino80p knockdown. Fig. S8. The transition of RNAPII in Ino80p knockdown is independent of both TSS usage and H2A.ZHtz1. Fig. S9. The Ino80 complex is essential for RNAPII pausing site determination associated with the + 1 nucleosome. Fig. S10. INO80 knockdown yields RNAPII pausing site determination defect in mESCs. Table S1. Summary of PRO-seq reads and reproducibility obtained in this study. Table S2. List of S. cerevisiae strains used in this study

Kinetic data of DUSP26-N for catalysis of DiFMUP.

<p>Kinetic data of DUSP26-N for catalysis of DiFMUP.</p

Structural comparison between DUSP26-N (C152S) and other DUSPs.

<p>Structural superposition of DUSP27 (magenta) (A), VHR (violet) (B), VH1 (blue) (C), and MKP-4 (gray) (D) onto DUSP26-N (C152S) (green). The large structural difference between the β3-α4 loop of DUSP26-N (C152S) and the corresponding loops of other DUSPs is indicated by black arrows. A red dot indicates the location of the C_α atom of the catalytic cysteine.</p

Comparison of the PTP-loop conformation between DUSP26-N (C152S) and other DUSPs.

<p>The PTP-loop conformation of DUSP26-N (C152S) (A), DUSP26-C (PDB code: 2E0T) (B), VHR (PDB code: 1VHR) (C), and MKP-4 (PDB code: 3LJ8) (D) are shown. The red arrows highlight the backbone amide atoms that are flipped away from the active site.</p

Overall structure of DUSP26-N (C152S).

<p>(A) A ribbon model of the monomeric DUSP26-N (C152S) structure. Secondary structural elements are labeled. (B) A ribbon model of the dimeric DUSP26-N (C152S) structure. Chains A and B of DUSP26-N (C152S) are colored blue and green, respectively. The Ser152 residue is shown as a red dot. The N- and C-termini are labeled.</p

Phosphatase activity of DUSP26-N.

<p>(A) Sequence alignment of DUSP26 with DUSP27, VHR, and VH1 in the N-terminal region. The DUSP26-N (C152S) and DUSP26-C (C152S) constructs are shown and the location of the α1-helix in DUSP27 is indicated. The identical residues and homologous residues are colored in dark and light blue, respectively. (B) Phosphatase activity measurements of DUSP26-N using DiFMUP as a substrate. Phosphatase activities of DUSP26-N wild-type (WT) (green), DUSP26-N (C152S) (red), and DUSP26-C WT (blue) were determined by monitoring the fluorescence emitted by the hydrolyzed DiFMU fluorogenic product.</p

Oligomerization state of DUSP26-N (C152S) in solution as determined by SEC-MALS.

<p>The molecular mass of DUSP26-N (C152S) was calculated from the elution profile examined by analytical SEC-MALS. The molecular weight determined by SEC-MALS corresponds to a monomer of DUSP26-N (C152S). The corresponding theoretical mass of DUSP26-N (C152S) is 20,000 Da.</p

Critical role of the N-terminal region in the catalytic activity of DUSP26-N.

<p>(A) Superposition of the DUSP26-C monomer (red) onto the DUSP26-N (C152S) monomer (green). The black dotted circle highlights the large conformational difference in the α7-α8 loop between DUSP26-N (C152S) and DUSP26-C. The additional N-terminal region (residues 39–60) of DUSP26-N (C152S) is colored blue. (B) Hydrogen bonding interaction network among the PTP-loop, its surrounding α7-α8 loop, and α1-helix in DUSP26-N (C152S). Residues in the PTP-loop (Gly155, Val156, and Ser157) and the N-terminal domain (Arg50, Tyr53, and Lys56) form hydrogen bonds with residues in the α7-α8 loop. The residues in the N-terminal domain, α7-α8 loop, and PTP-loop are shown as light blue, green, and dark blue, respectively. Hydrogen bonds are indicated by dotted lines.</p

Data-collection and refinement statistics.

<p>Data-collection and refinement statistics.</p