Intratumor molecular heterogeneity of hepatocellular carcinoma is partly attributed to the presence of hepatic cancer stem cells (CSCs). Different CSC populations defined by various cell surface ...markers may contain different oncogenic drivers, posing a challenge in defining molecularly targeted therapeutics. We combined transcriptomic and functional analyses of hepatocellular carcinoma cells at the single‐cell level to assess the degree of CSC heterogeneity. We provide evidence that hepatic CSCs at the single‐cell level are phenotypically, functionally, and transcriptomically heterogeneous. We found that different CSC subpopulations contain distinct molecular signatures. Interestingly, distinct genes within different CSC subpopulations are independently associated with hepatocellular carcinoma prognosis, suggesting that a diverse hepatic CSC transcriptome affects intratumor heterogeneity and tumor progression. Conclusion: Our work provides unique perspectives into the biodiversity of CSC subpopulations, whose molecular heterogeneity further highlights their role in tumor heterogeneity, prognosis, and hepatic CSC therapy. (Hepatology 2018;68:127‐140).
The dynamic nature of the chromatin epigenetic landscape plays a key role in the establishment and maintenance of cell identity, yet the factors that affect the dynamics of the epigenome are not ...fully known. Here we find that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 preferentially colocalize with epigenetic marks of active chromatin, and with cell-type specific enhancers. Loss of HMGNs enhances the rate of OSKM induced reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs), and the ASCL1 induced conversion of fibroblast into neurons. During transcription factor induced reprogramming to pluripotency, loss of HMGNs accelerates the erasure of the MEF-specific epigenetic landscape and the establishment of an iPSCs-specific chromatin landscape, without affecting the pluripotency potential and the differentiation potential of the reprogrammed cells. Thus, HMGN proteins modulate the plasticity of the chromatin epigenetic landscape thereby stabilizing, rather than determining cell identity.
During V(D)J recombination, RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) that contain either 12- or 23-nt spacer regions. Coordinated 12/23 cleavage ...predicts that DSBs at variable (V) gene segments should equal the level of breakage at joining (J) segments. Contrary to this, here we report abundant RAG-dependent DSBs at multiple Vκ gene segments independent of V-J rearrangement. We find that a large fraction of Vκ gene segments are flanked not only by a bone-fide 12 spacer but also an overlapping, 23-spacer flipped RSS. These compatible pairs of RSSs mediate recombination and deletion inside the Vκ cluster even in the complete absence of Jκ gene segments and support a V(D)J recombination center (RC) independent of the conventional Jκ-centered RC. We propose an improved model of Vκ-Jκ repertoire formation by incorporating these surprisingly frequent, evolutionarily conserved intra-Vκ cluster recombination events.
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•Vκ gene segments rearrange within the Vκ cluster independent of Jκ gene segments•Intra-Vκ recombination is mediated by flipped RSS embedded in bona fide RSS•An independent RAG recombination center assembles in the Vκ cluster•Intra-Vκ cluster recombination influences ultimate Vκ gene segment usage
Shinoda et al. demonstrate previously unknown, frequent RAG-dependent recombination within the Ig Vκ gene cluster, which is mediated by evolutionarily conserved flipped RSS embedded in bona fide RSS. These intra-Vκ cluster recombination events alter the available Vκ gene pool by deleting Vκ segments and shape the Vκ-Jκ immune repertoire.
IFN-regulatory factor 5 (IRF-5), a member of the IRF family, is a transcription factor that has a key role in the induction of the antiviral and inflammatory response. When compared with C57BL/6 ...mice, Irf5⁻/⁻ mice show higher susceptibility to viral infection and decreased serum levels of type I IFN and the inflammatory cytokines IL-6 and TNF-α. Here, we demonstrate that IRF-5 is involved in B-cell maturation and the stimulation of Blimp-1 expression. The Irf5⁻/⁻ mice develop an age-related splenomegaly, associated with a dramatic accumulation of CD19⁺B220⁻ B cells and a disruption of normal splenic architecture. Splenic B cells from Irf5⁻/⁻ mice also exhibited a decreased level of plasma cells. The CD19⁺ Irf5⁻/⁻ B cells show a defect in Toll-like receptor (TLR) 7- and TLR9-induced IL-6 production, and the aged Irf5⁻/⁻ mice have decreased serum levels of natural antibodies; however, the antigen-specific IgG1 primary response was already dependent in IRF-5 in young mice, although the IgM response was not. Analysis of the profile of transcription factors associated with plasma cell differentiation shows down-regulation of Blimp-1 expression, a master regulator of plasma cell differentiation, which can be reconstituted with ectopic IRF-5. IRF-5 stimulates transcription of the Prdm1 gene encoding Blimp-1 and binds to the IRF site in the Prdm1 promoter. Collectively, these results reveal that the age-related splenomegaly in Irf5⁻/⁻ mice is associated with an accumulation of CD19⁺B220⁻ B cells with impaired functions and show the role of IRF-5 in the direct regulation of the plasma cell commitment factor Blimp-1 and in B-cell terminal differentiation.
CD47 regulates the trafficking of specific coding and noncoding RNAs into extracellular vesicles (EVs), and the RNA contents of CD47+ EVs differ from that of CD63+ EVs released by the same cells. ...Single particle interferometric reflectance imaging sensing combined with immunofluorescent imaging was used to analyse the colocalization of tetraspanins, integrins, and CD47 on EVs produced by wild type and CD47‐deficient Jurkat T lymphoblast and PC3 prostate carcinoma cell lines. On Jurkat cell‐derived EVs, β1 and α4 integrin subunits colocalized predominantly with CD47 and CD81 but not with CD63 and CD9, conserving the known lateral interactions between these proteins in the plasma membrane. Although PC3 cell‐derived EVs lacked detectable α4 integrin, specific association of CD81 with β1 and CD47 was preserved. Loss of CD47 expression in Jurkat cells significantly reduced β1 and α4 levels on EVs produced by these cells while elevating CD9+, CD63+, and CD81+ EVs. In contrast, loss of CD47 in PC3 cells decreased the abundance of CD63+ and CD81+ EVs. These data establish that CD47+ EVs are mostly distinct from EVs bearing the tetraspanins CD63 and CD9, but CD47 also indirectly regulates the abundance of EVs bearing these non‐interacting tetraspanins via mechanisms that remain to be determined.
Bax, a pro‐apoptotic protein from the Bcl‐2 family, is central to apoptosis regulation. To suppress spontaneous apoptosis, Bax must be under stringent control that may include regulation of Bax ...conformation and expression levels. We report that IBRDC2, an IBR‐type RING‐finger E3 ubiquitin ligase, regulates the levels of Bax and protects cells from unprompted Bax activation and cell death. Downregulation of IBRDC2 induces increased cellular levels and accumulation of the active form of Bax. The ubiquitination‐dependent regulation of Bax stability is suppressed by IBRDC2 downregulation and stimulated by IBRDC2 overexpression in both healthy and apoptotic cells. Although mostly cytosolic in healthy cells, upon induction of apoptosis, IBRDC2 accumulates in mitochondrial domains enriched with Bax. Mitochondrial accumulation of IBRDC2 occurs in parallel with Bax activation and also depends on the expression levels of Bcl‐xL. Furthermore, IBRDC2 physically interacts with activated Bax. By applying Bax mutants in HCT116 Bax−/− cells, combined with the use of active Bax‐specific antibodies, we have established that both mitochondrial localization and apoptotic activation of Bax are required for IBRDC2 translocation to the mitochondria.
Background:
Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal cancer. Aberrant microRNA (miR) expression has been linked to carcinogenesis; however, no ...reports document a relationship between IBD‐related neoplasia (IBDN) and altered miR expression. In the current study we sought to identify specific miR dysregulation along the normal–inflammation–cancer axis.
Methods:
miR microarrays and quantitative reverse‐transcriptase polymerase chain reaction (RT‐PCR) were used to detect dysregulated miRs. Receiver operating characteristic curve analysis was employed to test for potential usefulness of miR‐31 as a disease marker of IBDNs. In silico prediction analysis, Western blot, and luciferase activity measurement were employed for target identification.
Results:
Several dysregulated miRs were identified between chronically inflamed mucosae and dysplasia arising in IBD. MiR‐31 expression increases in a stepwise fashion during progression from normal to IBD to IBDN and accurately discriminated IBDNs from normal or chronically inflamed tissues in IBD patients. Finally, we identified factor inhibiting hypoxia inducible factor 1 as a direct target of miR‐31.
Conclusions:
Our study reveals specific miR dysregulation as chronic inflammation progresses to dysplasia. MiR‐31 expression levels increase with disease progression and accurately discriminates between distinct pathological entities that coexist in IBD patients. The novel effect of miR‐31 on regulating factor inhibiting hypoxia inducible factor 1 expression provides a new insight on the pathogenesis of IBDN. (Inflamm Bowel Dis 2011;)
Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the ...recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX-/-mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNA-repair complexes on damaged DNA.
53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair ...(Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for "classic" nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.
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