The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome, resulting in altered patterns of gene ...expression. Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs) that occurs upon derivation of human iPSCs and identify numerous lincRNAs whose expression is linked to pluripotency. Among these, we defined ten lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells, suggesting that their activation may promote the emergence of iPSCs. Supporting this, our results indicate that these lincRNAs are direct targets of key pluripotency transcription factors. Using loss-of-function and gain-of-function approaches, we found that one such lincRNA (lincRNA-RoR) modulates reprogramming, thus providing a first demonstration for critical functions of lincRNAs in the derivation of pluripotent stem cells.
Human dermal fibroblasts obtained by skin biopsy can be reprogrammed directly to pluripotency by the ectopic expression of defined transcription factors. Here, we describe the derivation of induced ...pluripotent stem cells from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4/SOX2/KLF4/MYC. Blood-derived human induced pluripotent stem cells are indistinguishable from human embryonic stem cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes, and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human blood will allow the generation of patient-specific stem cells for diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.
Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, ...it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use.
Embryonic stem (ES) cells are pluripotent cells with the ability to self-renew indefinitely. These unique properties are controlled by genetic factors and chromatin structure. The exit from the ...self-renewing state is accompanied by changes in epigenetic chromatin modifications such as an induction in the silencing-associated histone H3 Lys 9 dimethylation and trimethylation (H3K9Me2/Me3) marks. Here, we show that the H3K9Me2 and H3K9Me3 demethylase genes, Jmjd1a and Jmjd2c, are positively regulated by the ES cell transcription factor Oct4. Interestingly, Jmjd1a or Jmjd2c depletion leads to ES cell differentiation, which is accompanied by a reduction in the expression of ES cell-specific genes and an induction of lineage marker genes. Jmjd1a demethylates H3K9Me2 at the promoter regions of Tcl1, Tcfcp2l1, and Zfp57 and positively regulates the expression of these pluripotency-associated genes. Jmjd2c acts as a positive regulator for Nanog, which encodes for a key transcription factor for self-renewal in ES cells. We further demonstrate that Jmjd2c is required to reverse the H3K9Me3 marks at the Nanog promoter region and consequently prevents transcriptional repressors HP1 and KAP1 from binding. Our results connect the ES cell transcription circuitry to chromatin modulation through H3K9 demethylation in pluripotent cells.
We compared bona fide human induced pluripotent stem cells (iPSCs) derived from umbilical cord blood (CB) cells and neonatal keratinocytes (K). As a consequence of both incomplete erasure of ...tissue-specific methylation and aberrant de novo methylation, CB-iPSCs and K-iPSCs were distinct in genome-wide DNA methylation profiles and differentiation potential. Extended passage of some iPSC clones in culture did not improve their epigenetic resemblance to embryonic stem cells, implying that some human iPSCs retain a residual 'epigenetic memory' of their tissue of origin.
H3.3 is a histone variant, which is deposited on genebodies and regulatory elements, by Hira, marking active transcription. Moreover, H3.3 is deposited on heterochromatin by Atrx/Daxx complex. The ...exact role of H3.3 in cell fate transition remains elusive. Here, we investigate the dynamic changes in the deposition of the histone variant H3.3 during cellular reprogramming. H3.3 maintains the identities of the parental cells during reprogramming as its removal at early time-point enhances the efficiency of the process. We find that H3.3 plays a similar role in transdifferentiation to hematopoietic progenitors and neuronal differentiation from embryonic stem cells. Contrastingly, H3.3 deposition on genes associated with the newly reprogrammed lineage is essential as its depletion at the later phase abolishes the process. Mechanistically, H3.3 deposition by Hira, and its K4 and K36 modifications are central to the role of H3.3 in cell fate conversion. Finally, H3.3 safeguards fibroblast lineage by regulating Mapk cascade and collagen synthesis.
Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil ...states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1
state. Reprogrammed dcTRAIL-R1
neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.
•KRAB-ZFPs-Trim28 centered canonical pathway for regulation of provirus and endogenous retroviruses (ERVs).•Non-canonical pathways mediating ERVs.•Important roles of ERVs in maintaining stemness and ...cell fate reprogramming.•Co-evolutionary model of ERVs and Kruppel associated box-Zinc Finger Proteins (KRAB-ZFP).
Recent advances in our understanding of endogenous retroviruses (ERVs) regulation and its functional aspects have provided us with vast power to unravel its role in the host's genome. Co-evolutionary model of ERVs and Kruppel associated box-Zinc Finger Proteins (KRAB-ZFPs) provides a deeper knowledge of how the genome is shaped during the course of evolution. However, the role of ERVs in normal cellular function still remains an enigma. Here we review studies in recent years with a focus on the role of ERVs in maintaining stemness and cell fate reprogramming, along with the recent discoveries of novel regulatory factors which have been shown to mediate ERV expression in both canonical and non-canonical pathways.
Embryonic stem cells (ESCs) repress the expression of exogenous proviruses and endogenous retroviruses (ERVs). Here, we systematically dissected the cellular factors involved in provirus repression ...in embryonic carcinomas (ECs) and ESCs by a genome-wide siRNA screen. Histone chaperones (Chaf1a/b), sumoylation factors (Sumo2/Ube2i/Sae1/Uba2/Senp6), and chromatin modifiers (Trim28/Eset/Atf7ip) are key determinants that establish provirus silencing. RNA-seq analysis uncovered the roles of Chaf1a/b and sumoylation modifiers in the repression of ERVs. ChIP-seq analysis demonstrates direct recruitment of Chaf1a and Sumo2 to ERVs. Chaf1a reinforces transcriptional repression via its interaction with members of the NuRD complex (Kdm1a, Hdac1/2) and Eset, while Sumo2 orchestrates the provirus repressive function of the canonical Zfp809/Trim28/Eset machinery by sumoylation of Trim28. Our study reports a genome-wide atlas of functional nodes that mediate proviral silencing in ESCs and illuminates the comprehensive, interconnected, and multi-layered genetic and epigenetic mechanisms by which ESCs repress retroviruses within the genome.
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•Genome-wide siRNA screen identifies key determinants for proviral silencing in ESCs•Histone chaperones, sumoylation factors, and chromatin modifiers can repress ERVs•Sumo2 orchestrates viral silencing through sumoylation modification of Trim28•Chaf1a regulates provirus and ERVs via its interaction with Eset, Kdm1a, and Hdac1/2
Proviral silencing is a characteristic of the pluripotent state, and the precise expression of endogenous retrovirus is critical for embryogenesis and development. In this study, a genome-wide siRNA screen identifies new cellular factors and new mechanisms involved in retroviral repression in embryonic stem cells.
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