[Show abstract][Hide abstract] ABSTRACT: Background: Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into every cell type and to self-renew. These characteristics correlate with a distinct nuclear architecture, epigenetic signatures enriched for active chromatin marks and hyperdynamic binding of structural chromatin proteins. Recently, several chromatin-related proteins have been shown to regulate ESC pluripotency and/or differentiation, yet the role of the major heterochromatin proteins in pluripotency is unknown. Results: Here we identify Heterochromatin Protein 1β (HP1β) as an essential protein for proper differentiation, and, unexpectedly, for the maintenance of pluripotency in ESCs. In pluripotent and differentiated cells HP1β is differentially localized and differentially associated with chromatin. Deletion of HP1β, but not HP1aα, in ESCs provokes a loss of the morphological and proliferative characteristics of embryonic pluripotent cells, reduces expression of pluripotency factors and causes aberrant differentiation. However, in differentiated cells, loss of HP1β has the opposite effect, perturbing maintenance of the differentiation state and facilitating reprogramming to an induced pluripotent state. Microscopy, biochemical fractionation and chromatin immunoprecipitation reveal a diffuse nucleoplasmic distribution, weak association with chromatin and high expression levels for HP1β in ESCs. The minor fraction of HP1β that is chromatin-bound in ESCs is enriched within exons, unlike the situation in differentiated cells, where it binds heterochromatic satellite repeats and chromocenters. Conclusions: We demonstrate an unexpected duality in the role of HP1β: it is essential in ESCs for maintaining pluripotency, while it is required for proper differentiation in differentiated cells. Thus, HP1β function both depends on, and regulates, the pluripotent state.
[Show abstract][Hide abstract] ABSTRACT: Diverting a protein from its intracellular location is a unique property of intrabodies. To interfere with the intracellular traffic of heterochromatin protein 1β (HP1β) in living cells, we have generated a cytoplasmic targeted anti-HP1β intrabody, specifically directed against the C-terminal portion of the molecule. HP1β is a conserved component of mouse and human constitutive heterochromatin involved in diverse nuclear functions including gene silencing, DNA repair and nuclear membrane assembly. We found that the anti-HP1β intrabody sequesters HP1β into cytoplasmic aggregates, inhibiting its traffic to the nucleus. Lamin B receptor (LBR) and a subset of core histones (H3/H4) are also specifically co-sequestered in the cytoplasm of anti-HP1β intrabody-expressing cells. Methylated histone H3 at K9 (Me9H3), a marker of constitutive heterochromatin, is not affected by the anti-HP1β intrabody expression. Hyper-acetylating conditions completely dislodge H3 from HP1βːLBR containing aggregates. The expression of anti-HP1β scFv fragments induces apoptosis, associated with an alteration of nuclear morphology. Both these phenotypes are specifically rescued either by overexpression of recombinant full length HP1β or by HP1β mutant containing the chromoshadow domain, but not by recombinant LBR protein. The HP1β-chromodomain mutant, on the other hand, does not rescue the phenotypes, but does compete with LBR for binding to HP1β⊡ These findings provide new insights into the mode of action of cytoplasmic-targeted intrabodies and the interaction between HP1β and its binding partners involved in peripheral heterochromatin organization.
No preview · Article · Sep 2015 · Experimental Cell Research
[Show abstract][Hide abstract] ABSTRACT: Mammals have three HP1 protein isotypes HP1 beta (CBX1), HP1 alpha (CBX3) and HP1 alpha (CBX5) that are encoded by the corresponding genes Cbx1, Cbx3 and Cbx5. Recent work has shown that reduction of CBX3 protein in homozygotes for a hypomorphic allele (Cbx3hypo) causes a severe postnatal mortality with around 99 percent of the homozygotes dying before weaning. It is not known what the causes of the postnatal mortality are. Here we show that Cbx3hypo/hypo conceptuses are significantly reduced in size and the placentas exhibit a haplo-insufficiency. Late gestation Cbx3hypo/hypo placentas have reduced mRNA transcripts for genes involved in growth regulation, amino acid and glucose transport. Blood vessels within the Cbx3hypo/hypo placental labyrinth are narrower than wild-type. Newborn Cbx3hypo/hypo pups are hypoglycemic, the livers are depleted of glycogen reserves and there is almost complete loss of stored lipid in brown adipose tissue (BAT). There is a 10-fold reduction in expression of the BAT-specific Ucp1 gene, whose product is responsible for nonshivering themogenesis. We suggest that it is the small size of the Cbx3hypo/hypo neonates, a likely consequence of placental growth and transport defects, combined with a possible inability to thermoregulate that causes the severe postnatal mortality.
No preview · Article · Jun 2015 · Journal of Biosciences
[Show abstract][Hide abstract] ABSTRACT: Mammals have three HP1 protein isotypes HP1β (CBX1), HP1γ (CBX3) and HP1α (CBX5) that are encoded by the corresponding genes Cbx1, Cbx3 and Cbx5. Recent work has shown that reduction of CBX3 protein in homozygotes for a hypomorphic allele (Cbx3 hypo) causes a severe postnatal mortality with around 99% of the homozygotes dying before weaning. It is not known what the causes of the postnatal mortality are. Here we show that Cbx3 hypo/hypo conceptuses are significantly reduced in size and the placentas exhibit a haplo-insufficiency. Late gestation Cbx3 hypo/hypo placentas have reduced mRNA transcripts for genes involved in growth regulation, amino acid and glucose transport. Blood vessels within the Cbx3 hypo/hypo placental labyrinth are narrower than wild-type. Newborn Cbx3 hypo/hypo pups are hypoglycemic, the livers are depleted of glycogen reserves and there is almost complete loss of stored lipid in brown adipose tissue (BAT). There is a 10-fold reduction in expression of the BAT-specific Ucp1 gene, whose product is responsible for non-shivering themogenesis. We suggest that it is the small size of the Cbx3 hypo/hypo neonates, a likely consequence of placental growth and transport defects, combined with a possible inability to thermoregulate that causes the severe postnatal mortality.
No preview · Article · Jun 2015 · Journal of Biosciences
[Show abstract][Hide abstract] ABSTRACT: We measured the dynamics of an essential epigenetic modifier, HP1β, in human cells at different stages of differentiation using Fluorescence Recovery After Photobleaching (FRAP). We found that HP1β mobility is similar in human embryonic stem cells (hES) and iPS cells where it is more mobile compared to fibroblasts; HP1β is less mobile in senescent fibroblasts than in young (dividing) fibroblasts. Introduction of "reprogramming factors", Oct4, Sox2, Klf4, cMyc and Lin28, into senescent fibroblasts and measuring the changes in HP1β mobility as reprogramming proceeds shows that the mobility of HP1β in senescent cells increases and by day 9 is the same as that found in young fibroblasts. Thus the dynamics of a key epigenetic modifier can be rejuvenated without de-differentiation through an embryonic stage. Future work will test whether other aspects of cellular physiology that age can be so rejuvenated without de-differentiation.
[Show abstract][Hide abstract] ABSTRACT: Background
In mouse embryonic stem cells (mESCs), transcriptional silencing of numerous class I and II endogenous retroviruses (ERVs), including IAP, ETn and MMERVK10C, is dependent upon the H3K9 methyltransferase (KMTase) SETDB1/ESET and its binding partner KAP1/TRIM28. In contrast, the H3K9 KMTases G9a and GLP and HP1 proteins are dispensable for this process. Intriguingly, MERVL retroelements are actively transcribed exclusively in the two-cell (2C) embryo, but the molecular basis of silencing of these class III ERVs at later developmental stages has not been systematically addressed.
Here, we characterized the roles of these chromatin factors in MERVL silencing in mESCs. While MMERVK10C and IAP ERVs are bound by SETDB1 and KAP1 and are induced following their deletion, MERVL ERVs show relatively low levels of SETDB1 and KAP1 binding and are upregulated exclusively following KAP1 depletion, indicating that KAP1 influences MERVL expression independent of SETDB1. In contrast to class I and class II ERVs, MERVL and MERVL LTR-driven genic transcripts are also upregulated following depletion of G9a or GLP, and G9a binds directly to these ERVs. Consistent with a direct role for H3K9me2 in MERVL repression, these elements are highly enriched for G9a-dependent H3K9me2, and catalytically active G9a is required for silencing of MERVL LTR-driven transcripts. MERVL is also derepressed in HP1α and HP1β KO ESCs. However, like KAP1, HP1α and HP1β are only modestly enriched at MERVL relative to IAP LTRs. Intriguingly, as recently shown for KAP1, RYBP, LSD1 and G9a-deficient mESCs, many genes normally expressed in the 2C embryo are also induced in HP1 KO mESCs, revealing that aberrant expression of a subset of 2C-specific genes is a common feature in each of these KO lines.
Our results indicate that G9a and GLP, which are not required for silencing of class I and II ERVs, are recruited to MERVL elements and play a direct role in silencing of these class III ERVs, dependent upon G9a catalytic activity. In contrast, induction of MERVL expression in KAP1, HP1α and HP1β KO ESCs may occur predominantly as a consequence of indirect effects, in association with activation of a subset of 2C-specific genes.
Full-text · Article · Jun 2013 · Epigenetics & Chromatin
[Show abstract][Hide abstract] ABSTRACT: Induced pluripotent stem (iPS) cells have provided a rational means of obtaining histo-compatible tissues for 'patient-specific' regenerative therapies (Hanna et al. 2010; Yamanaka & Blau 2010). Despite the obvious potential of iPS cell-based therapies, there are certain problems that must be overcome before these therapies can become safe and routine (Ohi et al. 2011; Pera 2011). As an alternative, we have recently explored the possibility of using 'epigenetic rejuvenation', where the specialized functions of an old cell are rejuvenated in the absence of any change in its differentiated state (Singh & Zacouto 2010). The mechanism(s) that underpin 'epigenetic rejuvenation' are unknown and here we discuss model systems, using key epigenetic modifiers, which might shed light on the processes involved. Epigenetic rejuvenation has advantages over iPS cell techniques that are currently being pursued. First, the genetic and epigenetic abnormalities that arise through the cycle of dedifferentiation of somatic cells to iPS cells followed by redifferentiation of iPS cells into the desired cell type are avoided (Gore et al. 2011; Hussein et al. 2011; Pera 2011): epigenetic rejuvenation does not require passage through the de-/redifferentiation cycle. Second, because the aim of epigenetic rejuvenation is to ensure that the differentiated cell type retains its specialized function it makes redundant the question of transcriptional memory that is inimical to iPS cell-based therapies (Ohi et al. 2011). Third, to produce unrelated cell types using the iPS technology takes a long time, around three weeks, whereas epigenetic rejuvenation of old cells will take only a matter of days. Epigenetic rejuvenation provides the most safe, rapid and cheap route to successful regenerative medicine.
[Show abstract][Hide abstract] ABSTRACT: Figure S1. Derivation of Cbx5-/- mESCs via sequential targeted disruption of the Cbx5 gene. Figure S2. Derivation of Cbx1-/- mESCs via sequential targeted disruption of the Cbx1 gene. Figure S3. Profiling of trimethylated lysine 9 of histone 3 (H3K9me3) along the length of endogenous retroviruses (ERVs). Figure S4. Profiling of H3K9me3 and H4K20me3 in the sequence flanking ERVs in wild-type and Setdb1-knockout mESCs. Figure S5. Knockdown (KD) of Cdyl, Cdyl2, Chd4 or Mpp8 does not result in reactivation of proviral reporters. Figure S6. Simultaneous KD of Mpp8 and Cbx3 does not result in reactivation of the ERV reporters. Figure S7. Proviral reporters are modestly reactivated upon KD of H3K9me3-binding H3K4 demethylases Jarid1a-c. Figure S8. Proviral reporters are modestly reactivated upon KD of H3K9me3-binding SRA (SET- and RING-associated) domain proteins Uhrf1 and Uhrf2. Figure S9. The level of derepression of the ERV reporters is substantially reduced in the Setdb1-KD cells following KD of the H3K4 methyltransferase Wdr5. Table S1. Primers used in the study.
[Show abstract][Hide abstract] ABSTRACT: Endogenous retroviruses (ERVs) are parasitic sequences whose derepression is associated with cancer and genomic instability. Many ERV families are silenced in mouse embryonic stem cells (mESCs) via SETDB1-deposited trimethylated lysine 9 of histone 3 (H3K9me3), but the mechanism of H3K9me3-dependent repression remains unknown. Multiple proteins, including members of the heterochromatin protein 1 (HP1) family, bind H3K9me2/3 and are involved in transcriptional silencing in model organisms. In this work, we address the role of such H3K9me2/3 "readers" in the silencing of ERVs in mESCs.
We demonstrate that despite the reported function of HP1 proteins in H3K9me-dependent gene repression and the critical role of H3K9me3 in transcriptional silencing of class I and class II ERVs, the depletion of HP1α, HP1β and HP1γ, alone or in combination, is not sufficient for derepression of these elements in mESCs. While loss of HP1α or HP1β leads to modest defects in DNA methylation of ERVs or spreading of H4K20me3 into flanking genomic sequence, respectively, neither protein affects H3K9me3 or H4K20me3 in ERV bodies. Furthermore, using novel ERV reporter constructs targeted to a specific genomic site, we demonstrate that, relative to Setdb1, knockdown of the remaining known H3K9me3 readers expressed in mESCs, including Cdyl, Cdyl2, Cbx2, Cbx7, Mpp8, Uhrf1 and Jarid1a-c, leads to only modest proviral reactivation.
Taken together, these results reveal that each of the known H3K9me3-binding proteins is dispensable for SETDB1-mediated ERV silencing. We speculate that H3K9me3 might maintain ERVs in a silent state in mESCs by directly inhibiting deposition of active covalent histone marks.
[Show abstract][Hide abstract] ABSTRACT: Absence of HP1α and HP1β does not affect localization of H3K9me3. Immunofluorescence analysis of H3K9me3 in MEF cells derived from HP1αHP1β double knockout mice (HP1αβdn). DNA was visualized using DAPI. Bars, 7.5 µm.
[Show abstract][Hide abstract] ABSTRACT: H3 lysine 9 trimethylation (H3K9me3) is a histone posttranslational modification (PTM) that has emerged as hallmark of pericentromeric heterochromatin. This constitutive chromatin domain is composed of repetitive DNA elements, whose transcription is differentially regulated. Mammalian cells contain three HP1 proteins, HP1α, HP1β and HP1γ These have been shown to bind to H3K9me3 and are thought to mediate the effects of this histone PTM. However, the mechanisms of HP1 chromatin regulation and the exact functional role at pericentromeric heterochromatin are still unclear. Here, we identify activity-dependent neuroprotective protein (ADNP) as an H3K9me3 associated factor. We show that ADNP does not bind H3K9me3 directly, but that interaction is mediated by all three HP1 isoforms in vitro. However, in cells ADNP localization to areas of pericentromeric heterochromatin is only dependent on HP1α and HP1β. Besides a PGVLL sequence patch we uncovered an ARKS motif within the ADNP homeodomain involved in HP1 dependent H3K9me3 association and localization to pericentromeric heterochromatin. While knockdown of ADNP had no effect on HP1 distribution and heterochromatic histone and DNA modifications, we found ADNP silencing major satellite repeats. Our results identify a novel factor in the translation of H3K9me3 at pericentromeric heterochromatin that regulates transcription.
[Show abstract][Hide abstract] ABSTRACT: Absence of HP1α and HP1β does not affect ADNP, HP1γ or H3K9me3 levels. Western blot analysis of total cell extracts from wild type (wt) and HP1αHP1β double knockout (HP1αβdn) MEF cells using the indicated antibodies.
[Show abstract][Hide abstract] ABSTRACT: Localization of ADNP during the cell cycle. Immunofluorescence analysis of ADNP in NIH3T3 cells at interphase (A) and during M-phase (B). DNA was visualized using DAPI. Bars, 5 µm (A and B, lane 2) or 10 µm (B, rows 1, 3–4).
[Show abstract][Hide abstract] ABSTRACT: Expression levels of YFP-ADNP stable transfected cell lines. Western blot analysis of untransfected NIH3T3 cells or NIH3T3 cells stably expressing wild type YFP-ADNP (wt) or the indicated single or double mutant fusion proteins using the anti-ADNP antibody. The black arrowhead indicates the running position of endogenous ADNP; the open arrowhead indicates the running position of the YFP-ADNP fusion proteins.
[Show abstract][Hide abstract] ABSTRACT: Knockdown of ADNP does not affect DNA methylation levels. Genomic DNA of untreated (mock) and NIH3T3 cells transfected with scrambled (scr.) or ADNP targeting siRNAs as well as wild type (wt) and Suv39h1, Suv39h2 double knockout (Suv39h1/h2 dn) MEF cells was digested with the restriction enzyme TaiI and separated on a 1% agarose gel. Ethidium bromide staining of the gel (A) and Southern blot (B) using a major satellite repeat probe are shown.
[Show abstract][Hide abstract] ABSTRACT: Nuclear distribution of HP1α, HP1β and HP1γ is not affected by ADNP knockdown. Immunofluorescence analysis of HP1α (A), HP1β (B) and HP1γ (C) in wild type (wt) and Suv39h1, Suv39h2 double knockout (Suv39h1/h2 dn) MEF cells (top). Immunofluorescence analysis of HP1α (A), HP1β (B) and HP1γ (C) in untreated (mock) and NIH3T3 cells transfected with scrambled (scr.) or ADNP targeting siRNAs (bottom). DNA was visualized using DAPI. Bars, 5 µm.
[Show abstract][Hide abstract] ABSTRACT: Absence of single HP1 isoform proteins does not affect ADNP localization to pericentromeric heterochromatin. Immunofluorescence analysis of ADNP in wild type (wt) or mutant MEF cells of the indicated genomic background after knock out of the indicated HP1 genes. DNA was visualized using DAPI. Bars, 7.5 µm.
[Show abstract][Hide abstract] ABSTRACT: HP1 does not bind to ADNP K767me3. (A) The indicated ADNP and histone H3 peptides were used in pulldown experiments of HeLa S3 cell nuclear extract. Beads without coupled peptides were used as control (mock). Specifically recovered proteins were analyzed by western blotting using the indicated antibodies. (B) Fluorescence polarization binding assay of recombinant HP1β using the indicated H3 (unmodified, K9me3) and ADNP (unmodified, K767me3) peptides. The averaged fluorescence polarization signal from three independent titration reactions is shown.
[Show abstract][Hide abstract] ABSTRACT: Characterization of recombinant chromatin templates. (A) Agarose gel (1%) of free DNA template (12×200-601 DNA) and the indicated chromatin assembly reactions using either unmodified H3 or H3K9me3 stained with ethidium bromide. The running position of size standards (MW) is indicated on the left. (B) The indicated free DNA and chromatin assembled chromatin templates were digested with Mnase for the indicated time periods. Reactions were run on a 1% agarose gel and stained with ethidium bromide. (C) The indicated chromatin templates were run on an SDS PAGE gel and stained with Coomassie Blue. The running position of size standards (MW) is indicated on the left. (D) H3unmod. and H3K9me3 chromatin templates were analyzed by western blotting using the indicated antibodies.