XB-ART-54332Cold Spring Harb Symp Quant Biol 2017 Jan 01;82:1-14. doi: 10.1101/sqb.2017.82.033753.
Show Gene links Show Anatomy links
Shaping Chromatin in the Nucleus: The Bricks and the Architects.
Chromatin organization in the nucleus provides a vast repertoire of information in addition to that encoded genetically. Understanding how this organization impacts genome stability and influences cell fate and tumorigenesis is an area of rapid progress. Considering the nucleosome, the fundamental unit of chromatin structure, the study of histone variants (the bricks) and their selective loading by histone chaperones (the architects) is particularly informative. Here, we report recent advances in understanding how relationships between histone variants and their chaperones contribute to tumorigenesis using cell lines and Xenopus development as model systems. In addition to their role in histone deposition, we also document interactions between histone chaperones and other chromatin factors that govern higher-order structure and control DNA metabolism. We highlight how a fine-tuned assembly line of bricks (H3.3 and CENP-A) and architects (HIRA, HJURP, and DAXX) is key in adaptation to developmental and pathological changes. An example of this conceptual advance is the exquisite sensitivity displayed by p53-null tumor cells to modulation of HJURP, the histone chaperone for CENP-A (CenH3 variant). We discuss how these findings open avenues for novel therapeutic paradigms in cancer care.
PubMed ID: 29208640
Article link: Cold Spring Harb Symp Quant Biol
Species referenced: Xenopus
Genes referenced: cenpa daxx h3-3a h3-3b hira hras mst1 tp53 tubg1
GO keywords: DNA modification
Morpholinos: h3-3a MO3 hira MO1
Article Images: [+] show captions
|Figure 1. Epigenetic components of chromatin architecture and a complex network of interactions underlying cell fate determination. (A) Numerous epigenetic factors help organize chromatin in the 4D nucleus: along with the linker histone H1, DNAwrapped around one (H3–H4)2 tetramer capped by two H2A–H2B dimers forms the nucleosome—the fundamental repeating unit of chromatin. DNA can be methylated and histones can be posttranslationally modified (e.g., by methylation [Me], acetylation [Ac], and phosphorylation [P]). Chromatin-binding proteins such as methyl- or histone-recognizing factors read the information encoded by these covalent marks. The presence of histone variants adds further complexity. Arrays of nucleosomes fold into higher-order chromatin structures, potentially guided by noncoding RNA. The nuclear localization of a given chromosomal domain represents an additional level of regulatory information. (B) View from below of the historical Waddington landscape (top). Pegs represent genes and the strings represent a complex system of genetic interaction that determines cell fate. The epigenetic landscape is shaped by the strings, which are ultimately anchored to the genes. (Bottom) A modern re-interpretation of the Waddington landscape. The valleys represent ongoing decision events in cells (represented by colored beads) on their path to a final cell fate. (A, Adapted, with permission, from Probst et al. 2009; B, top, reprinted, with permission, from Waddington 1957; bottom, reproduced, with permission, from Paul L. Harrison and the Epigenesys Network.)|
|Figure 2. Histone variants (bricks) and their associated histone chaperones (architects) mark specific chromosomal regions. Among H3 variants in humans, at least four are handled by specific histone chaperones during particular cell cycle stages: new CENP-A, the centromeric variant, is incorporated at centromeres by HJURP during late mitosis–early G1; H3.1/2, the replicative variants, are incorporated genome-wide during DNA synthesis by CAF-1 complex; H3.3, the replacement form, is placed throughout the cell cycle at regulatory elements, gene bodies by the HIRA complex, and at telomeres and pericentric heterochromatin by the DAXX/ATRX complex.|
|Figure 3. DAXX mediates ectopic CENP-A deposition in chromosome arms. (A) Schematic representation of centromeric chromatin. Centromeres comprise CENP-A nucleosomes interspersed with H3.1/2 and H3.3 nucleosomes. Centromeric chromatin contains minor- (in mice) and α- (in humans) satellite repeats flanked by pericentric heterochromatin. (B) DNA FISH on metaphase spreads of p53-null mouse embryonic fibroblasts (MEFs) transduced with the indicated retroviral construct. Cells were stained with an antibody against CENP-A and LNA FISH (fluorescence in situ hybridization) probes for minor satellites (site of centromeric CENP-A deposition). Insets show individual magnified chromosomes. Overexpression (O/E) of CENPA, but not HJURP, leads to ectopic deposition of CENP-A on chromosome arms. (C) Fluorescent microscopy visualization of CENP-A after treatment with the indicated siRNA in a human cell line overexpressing CENP-A (HeLa eCENP-A). Depletion of histone chaperone DAXX, but not HJURP, rescues the ectopic CENP-A deposition phenotype. Histogram representing the fluorescence of CENP-A present in chromosome arms of metaphase spreads from two cell lines (wild-type, wtCENP-A and overexpression, eCENP-A) treated with the indicated siRNA. P values represent pairwise comparison of siLuc (control), and all other siRNAs tested with the same cell line with a Mann–Whitney U-test. (A, Adapted, with permission, from Muller and Almouzni 2017; B, adapted, with permission from Filipescu et al. 2017; C, adapted, with permission, from Lacoste et al. 2014.)|
|Figure 4. Cancer cells display addiction to chromatin architect HJURP. (A) Key chromatin factors, including important bricks and architects, display variations in expression during tumorigenesis. HJURP and CENP-A levels were assessed in experimentally transformed cells and in human cancer cells. Then, their levels were modified in cultured cells and in tumors to assess effects on tumorigenesis. (B) Gene expression levels of CENP-A and HJURP are higher in cancers with defective p53. Box plot comparisons of relative expression (mRNA) of genes coding for CENP-A (CENPA), HJURP (HJURP), and H3.1 (HIST1H3E) from all cancers (28 cancer types), classified according to p53 status (TCGA provisional data). Tumors are either wild type for TP53 (n = 4083) or p53 loss of function (LOF) (n = 257). Significance was computed using Wilcoxon rank sum tests. (C) HJURP or CENP-A overexpression alone does not transform p53-null MEFs. Proliferation curve of p53-null MEFs transduced with the indicated retroviral construct. The graph displays the quantified cell number ± SEM of triplicates. (D) HJURP and CENP-A are overexpressed in p53-null wild-type and transformed MEFs. Western blot of HJURP and CENP-A levels in RIPA-soluble extracts from MEFs transduced with empty vector or sequentially with E1A and HRas-V12. (E) Conditional depletion of exogenous HJURP stops the growth of established tumors lacking endogenous HJURP. Allograft assay measuring tumor growth following HJURP knockout over time. p53-null HRas-V12 MEFs were generated with an inducible CRISPR-resistant HJURP transgene maintained in the presence of doxycycline. Doxycycline was withdrawn on day 6 after injection (Hjurp switched off ) and allograft tumor volume was measured over time. Data represent mean tumor volume ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005, t-test. (Adapted, with permission, from Filipescu et al. 2017.)|
|Figure 5. H3.3 plays a nonredundant role in Xenopus early development. (A) Well-characterized Xenopus laevis embryonic development provides a crucial window for studying chromatin. The embryo undergoes rapid synchronous cell divisions without any zygotic transcription until the midblastula transition (MBT). After the MBT, gap phases are introduced in the cell cycle along with the onset of zygotic transcription. (B) Chromatin disorganization upon depletion of a crucial brick (H3.3) or architect (HIRA). MNase digestion profile of H3.3 and HIRA morpholino (MO) injected embryos. Stage 14 embryos were used to prepare nuclei. For each time point of MNase digestion, purified DNA fragments were analyzed by agarose gel electrophoresis. Densitometric profiles of the 1-min digestion products are shown on the right (Szenker et al. 2012). (C) H3.2 expression does not rescue loss of H3.3 during Xenopus early development. H3.3 MO and indicated mRNAs were injected into two-cell stage embryos. Stage 25 embryos were used for image acquisition. Percentage viability of 30 embryos is shown. (Adapted from Szenker et al. 2012.)|
|Figure 6. Dosage and context matter: important tenets of chromatin maintenance. (A) Overexpression of cenH3 results in partner switching within the chaperone–variant circuitry (Lacoste et al. 2014). Schematic model of cenH3 overexpression in human cells. In physiological conditions, cenH3-H4 is loaded at the centromere by HJURP. The H3.3–H4 pool is taken care of by DAXX or HIRA for loading in chromosome arms. When the pool of cenH3-H4 increases, HJURP is overexpressed and greater concentration of cenH3 is found at the centromere. The excess of cenH3 is handled by DAXX that loads or maintains it in chromosome arms. (B) Increasing levels of HJURP maintain tumorigenesis in cells lacking functional p53. The model integrates CENP-A deposition by HJURP with the activity of the p53 checkpoint pathway. There are different outcomes, following CENP-A or HJURP knockout, dependent on the p53 status. When cell cycle is slow and CENP-A levels are low in p53- proficient cells, genome integrity remains intact and is protected by the function of p53. Tumor growth arrest, due to loss of CENP-A at centromeres, in established p53-deficient tumors is striking and underlines a case of epigenetic-addiction to high levels of HJURP. If HJURP is depleted in this context, it leads to massive aneuploidy and apoptosis. Thus, tumors become addicted to the high level of HJURP in the absence of p53. (C) In Xenopus laevis, selective loss of H3.3 variant causes global chromatin dysregulation during early development (Szenker et al. 2012). Depletion of endogenous H3.3 impairs cellular viability leading to gastrulation defects. Histone chaperone HIRA knockdown gives rise to similar phenotypes, implicating the DNA synthesis-independent H3.3 incorporation pathway during development. (A, Adapted, with permission, from Lacoste et al. 2014; B, reprinted, with permission, from Filipescu et al. 2017.)|