Solution structural analysis of the single-domain parvulin TbPin1

10.1371/journal.pone.0043017PLoS ONE78

Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation

Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIAPET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated noncoding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells

Assigned ¹H-¹⁵N HSQC spectrum of TbPin1 in 20 mM sodium phosphate buffer at pH 7.0.

<p>The spectrum was recorded at 298 K on a Varian Unity Inova 600 MHz spectrometer. Resonance assignments of backbone amide groups are indicated by the residue type and number. The unlabeled resonances are the side chain amides. T25 and G72 are shown in negative resonances corresponding to the resonances aliased in the ¹⁵N dimension. Positive resonances are colored red and negative resonances are colored green.</p

Structural statistic for the 20 lowest energy structures of TbPin1.

a<p>Dihedral angle restraints are generated by TALOS+ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043017#pone.0043017-Shen1" target="_blank">[41]</a>.</p>b<p>Quality of the ensemble of the 20 lowest-energy structures of TbPin1 was assessed by PROCHECK-NMR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043017#pone.0043017-Doreleijers1" target="_blank">[61]</a>.</p

Chemical shift perturbation analysis reveals the substrate binding pocket on TbPin1.

<p>(A) Overlay of the ¹H-¹⁵N HSQC spectra of TbPin1 free (red) and in complex with the unlabeled phosphorylated peptide substrate (blue). The molar ratio of TbPin1 to the phosphorylated peptide is 1∶8. (B) Diagram of amide chemical shift changes (Δδ_HN) of TbPin1 versus residue number at a molar ratio of 1∶8. The average amide chemical shift change () and the mean standard deviation () are indicated with solid and dashed lines, respectively. Residues with chemical shift changes larger than (dashed line) are considered to be involved in substantial contact with the substrate. (C) Mapping the substantial contact residues on the tertiary structure of TbPin1. Residues with significant chemical shift change are highlighted in stick style.</p

Structural comparison of TbPin1 (2LJ4) with other parvulins.

<p>(A) hPin1 in complex with the Ala-Pro dipeptide and a sulfate ion (1PIN); (B) hPin1 without a sulfate ion (1F8A); (C) CaEss1 (1YW5); (D) Pin1At (1J6Y); (E) PrsA-PPIase domain (2JZV); (F) EcPar10 (1JNT). The structures of TbPin1, hPin1, CaEss1, Pin1At, EcPar10 and PrsA-PPIase domain are colored green, cyan, blue, pink, lightblue and yellow, respectively. The β1/α1 loop of TbPin1 is displayed in red. The sulfate ion is used to mimic to the phosphate group.</p

Model-free analysis of TbPin1.

<p>(A, C and E) The plots of the order parameters S², the effective internal correlation time τ_e, and the conformational exchange rate R_ex versus residue number of TbPin1, respectively; (B, D and F) Residues with S², τ_e (blue) and R_ex (orange) values are mapped onto the solution structure of TbPin1, respectively. (B) Gray, S² unavailable due to the absence of NMR data or failure in NMR data fitting; red, S²<0.6; orange, 0.6≤S²<0.7; yellow, 0.7≤S²<0.8; green, 0.8≤S²<0.9; and blue, 0.9≤S²<1.0. The ribbon graph was generated by PyMOL.</p

TbPin1 catalyzes the <i>cis-trans</i> isomerization of a phosphorylated peptide.

<p>A selected region of the 2D-EXSY spectrum of the phosphorylated peptide SSYFSG[p]TPLEDDSD is displayed (A) in the absence of TbPin1; (B) in the presence of TbPin1; (C) in the presence of the TbPin1-C65A mutant; (D) in the presence of the FHA-truncated TbPar42 mutant (TbPPIase). A mixing time of 300 ms was used. The <i>cis</i> and <i>trans</i> T7-HN and L9-HN are shown in the spectra, which were assigned based on the 2D-TOCSY spectrum of the peptide. Diagonal peaks from <i>cis</i> and <i>trans</i> conformers are indicated by cc and tt, respectively, whereas exchange peaks resulting from isomerization are labeled with ct and tc.</p

NanoVar : accurate characterization of patients' genomic structural variants using low-depth nanopore sequencing

The recent advent of third-generation sequencing technologies brings promise for better characterization of genomic structural variants by virtue of having longer reads. However, long-read applications are still constrained by their high sequencing error rates and low sequencing throughput. Here, we present NanoVar, an optimized structural variant caller utilizing low-depth (8X) whole-genome sequencing data generated by Oxford Nanopore Technologies. NanoVar exhibits higher structural variant calling accuracy when benchmarked against current tools using low-depth simulated datasets. In patient samples, we successfully validate structural variants characterized by NanoVar and uncover normal alternative sequences or alleles which are present in healthy individuals.Ministry of Education (MOE)National Research Foundation (NRF)Published versionWork in the T.B. laboratory is supported by the National Research Foundation, the Singapore Ministry of Education under its Centres of Excellence initiative and the RNA Biology Center at the Cancer Science Institute of Singapore, NUS, as part of funding under the Singapore Ministry of Education’s AcRF Tier 3 grants [MOE2014-T3-1-006]. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program. C.Y.T. and R.T.M. are supported by a Doctoral Scholarship from the Cancer Science Institute of Singapore

Super-Enhancers and Broad H3K4me3 Domains Form Complex Gene Regulatory Circuits Involving Chromatin Interactions

Stretched histone regions, such as super-enhancers and broad H3K4me3 domains, are associated with maintenance of cell identity and cancer. We connected super-enhancers and broad H3K4me3 domains in the K562 chronic myelogenous leukemia cell line as well as the MCF-7 breast cancer cell line with chromatin interactions. Super-enhancers and broad H3K4me3 domains showed higher association with chromatin interactions than their typical counterparts. Interestingly, we identified a subset of super-enhancers that overlap with broad H3K4me3 domains and show high association with cancer-associated genes including tumor suppressor genes. Besides cell lines, we could observe chromatin interactions by a Chromosome Conformation Capture (3C)-based method, in primary human samples. Several chromatin interactions involving super-enhancers and broad H3K4me3 domains are constitutive and can be found in both cancer and normal samples. Taken together, these results reveal a new layer of complexity in gene regulation by super-enhancers and broad H3K4me3 domains.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio