Regulation of the Boundaries of Accessible Chromatin

<div><p>Regulatory regions maintain nucleosome-depleted, open chromatin status but simultaneously require the presence of nucleosomes for specific histone modifications. It remains unclear how these can be achieved for proper regulatory function. Here we demonstrate that nucleosomes positioned within accessible chromatin regions near the boundaries provide platforms for histone modifications while preventing the occlusion of regulatory elements. These boundary nucleosomes were particularly enriched for active or poised regulatory marks in human, such as histone acetylations, H3K4 methylations, H3K9me3, H3K79me2, and H4K20me1. Additionally, we found that based on a genome-wide profiling of ∼100 recombinant yeast strains, the location of open chromatin borders tends to vary mostly within 150 bp upon genetic perturbation whereas this positional variation increases in proportion to the sequence preferences of the underlying DNA for nucleosome formation. More than 40% of the local boundary shifts were associated with genetic variation in <i>cis</i>- or <i>trans</i>-acting factors. A sizeable fraction of the identified genetic factors was also associated with nearby gene expression, which was correlated with the distance between the transcription start site (tss) and the boundary that faces the tss. Taken together, the variation in the width of accessible chromatin regions may arise in conjunction with the modulation of the boundary nucleosomes by post-translational modifications or by chromatin regulators and in association with the activity of nearby gene transcription.</p></div

Boundary nucleosome positioning within open chromatin.

<p>(A) Superposition of <i>in vivo</i> and <i>in vitro</i> nucleosomes surrounding the <i>in vivo</i> TFBSs (black curve) and sequence-predicted Transfac TFBSs (gray curve). (B) Number of DHS tags mapped to the region centered on the <i>in vivo</i> TFBSs (black curve) and sequence-predicted Transfac TFBSs (gray curve). (C) Chromatin structure in GM12878 at a genomic locus (chr2:232,378,500–232,379,800). Shown from top to bottom are tracks for open chromatin density (two replicates), chromatin states (red: active promoter, yellow: weak enhancer, and orange: strong enhancer), nucleosome density (blue box: boundary nucleosomes), histone modifications (density shown on the gray scale with dark indicating dense modifications), and TF binding locations (binding affinity shown on the same gray scale as above).</p

Local changes of yeast open chromatin upon genetic perturbation.

<p>(A) We identified 4,897 open chromatin loci in BY4716 and aligned them by the 5′ end, center, and 3′ end, and then mapped the relative locations of nearby open chromatin loci in the other 95 strains. The center is defined as the middle point between the 5′ and 3′ boundaries. The number of strains (0∼95) that matches its boundary or center within a given distance from the homologous boundary or center in BY4716 was obtained and the frequency of the overlappings is represented as color gradient according to the distance shown at the bottom of each heat map. The rows of each heat map correspond to each of the 4,897 chromatin sites in BY4716. (B) The average frequency of mapped locations as a function of the distance to the center or to the end of the homologous site in BY4716. (C) The average frequency of mapped locations according to the <i>in vitro</i> nucleosome score as a function of the distance to the center or to the end of the homologous site in BY4716.</p

Histone modifications and H2A.Z occupancy across open chromatin in human.

<p>Shown is the body of open chromatin, which is divided into ten bins, along with 1(A) Pattern of H3K27ac, H3K4me2, H3K4me3, and H3K9ac. (B) Pattern of H3K4me1, H3K79me2, H3K9me3, and H4K20me1. (C) Pattern of H3K27me3 and H3K36me3.</p

Positioning of boundary nucleosomes within open chromatin.

<p>(A) Superposition of <i>in vivo</i> and <i>in vitro</i> nucleosomes, and FAIRE read density across the boundaries of promoter-associated (left) or non-associated (right) open chromatin in yeast. (B) Superposition of <i>in vivo</i> and <i>in vitro</i> nucleosomes, and FAIRE read density across the boundaries of promoter-associated (left) or non-associated (right) open chromatin in human.</p

Oncogenic activation of the stat3 pathway drives pd-l1 expression in natural killer/t-cell lymphoma

Mature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.ASTAR (Agency for Sci., Tech. and Research, S’pore)NMRC (Natl Medical Research Council, S’pore)MOH (Min. of Health, S’pore

Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-cell lymphoma

10.1182/blood-2018-01-829424BLOOD132111146-115

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters - Fluke-Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3'UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores - mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer