CAF-1 Is Essential for Heterochromatin Organization in Pluripotent Embryonic Cells

During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells

Histone H3 trimethylation at lysine 36 is associated with constitutive and facultative heterochromatin

The mammalian genome contains numerous regions known as facultative heterochromatin, which contribute to transcriptional silencing during development and cell differentiation. We have analyzed the pattern of histone modifications associated with facultative heterochromatin within the mouse imprinted Snurf–Snrpn cluster, which is homologous to the human Prader-Willi syndrome genomic region. We show here that the maternally inherited Snurf–Snrpn 3-Mb region, which is silenced by a potent transcription repressive mechanism, is uniformly enriched in histone methylation marks usually found in constitutive heterochromatin, such as H4K20me3, H3K9me3, and H3K79me3. Strikingly, we found that trimethylated histone H3 at lysine 36 (H3K36me3), which was previously identified as a hallmark of actively transcribed regions, is deposited onto the silenced, maternally contributed 3-Mb imprinted region. We show that H3K36me3 deposition within this large heterochromatin domain does not correlate with transcription events, suggesting the existence of an alternative pathway for the deposition of this histone modification. In addition, we demonstrate that H3K36me3 is markedly enriched at the level of pericentromeric heterochromatin in mouse embryonic stem cells and fibroblasts. This result indicates that H3K36me3 is associated with both facultative and constitutive heterochromatin. Our data suggest that H3K36me3 function is not restricted to actively transcribed regions only and may contribute to the composition of heterochromatin, in combination with other histone modifications

Acetylation is important for MyoD function in adult mouse

Acetylation is a post-translational modification that influences the activity of numerous proteins in vitro. Among them, the myogenic transcription factor MyoD displays an increased transcriptional activity in vitro when acetylated on two lysines, lysine 99 and 102. Here, we have investigated the biological relevance of this acetylation in vivo. Using specific antibodies, we demonstrate that endogenous MyoD is acetylated on lysine 99 and 102 in myoblasts. Moreover, we show the functional importance of acetylation in live animals, using a mutant of MyoD in which lysines 99 and 102 were replaced by arginines. Knock-in (KI) embryos homozygous for the MyoD R 99,102 allele expressed slightly reduced levels of MyoD, but they developed normally. However, the KI homozygous adult mice showed a phenotype close to that of MyoD knock-out animals, including delayed muscle regeneration in vivo, and increased numbers of myoblasts but with reduced differentiation potential in vitro. Taken together, these results demonstrate the importance of MyoD acetylation for adult myogenesis

Acetylation is important for MyoD function in adult mice

Acetylation is a post-translational modification that influences the activity of numerous proteins in vitro. Among them, the myogenic transcription factor MyoD shows an increased transcriptional activity in vitro when acetylated on two lysines (K): lysines 99 and 102. Here, we have investigated the biological relevance of this acetylation in vivo. Using specific antibodies, we show that endogenous MyoD is acetylated on lysines 99 and 102 in myoblasts. Moreover, we show the functional importance of acetylation in live animals by using a mutant of MyoD in which lysines 99 and 102 were replaced by arginines (R). Knock-in embryos homozygous for the MyoD(R99,102) allele expressed slightly reduced levels of MyoD but developed normally. However, the knock-in homozygous adult mice showed a phenotype that was almost identical to that of MyoD-knockout animals, including delayed muscle regeneration in vivo and an increased number of myoblasts but with reduced differentiation potential in vitro. Together, these results show the importance of MyoD acetylation for adult myogenesis

Elastin in silico.

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Nucleosomal Organization Is Not Altered in p150CAF-1-Depleted ES Cells

<div><p>Nuclei were prepared from ES cells transfected with control or p150CAF-1 siRNA vector. Nuclei were digested with increasing amounts of DNase I or MNase. (A) After digestion with the indicated nucleases, total DNA was prepared and run onto an agarose gel which was stained with ethidium bromide to reveal bulk genomic DNA.</p><p>(B) The DNA was blotted onto a nylon membrane, which was then hybridized with the α-³²P-labeled pSAT major satellite repeat probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b047" target="_blank">47</a>].</p></div

Depletion of p150CAF-1 in ES Cells Results in a Severe Alteration of Heterochromatin Organization

<div><p>(A) Strategy used to deplete p150CAF-1 by RNAi in ES cells. The siRNA expression vector includes a puromycin selection cassette (Puro), the mouse H1 promoter, and the siRNA encoding sequence. ES cells were kept under puromycin selection during 48 h following transfection.</p><p>(B) Abnormal heterochromatin organization in p150CAF-1-depleted ES cells. Immunodetection of p150CAF-1 (green) and HP1α (red) in ES cells transfected with control (cont) and p150CAF-1 siRNA. siRNA expression results in efficient p150CAF-1 depletion. The right-hand image shows the merge between HP1α fluorescence and DAPI-stained DNA in blue. Scale bar = 10 μm.</p><p>(C) Heterochromatin organization is not altered in p150CAF-1-depleted MEFs. Immunodetection of p150CAF-1 (green) and HP1α (red) in MEFs transfected with control (cont) or p150CAF-1 siRNA. The right-hand image shows the merge between HP1α fluorescence and DAPI-stained DNA in blue.</p><p>(D) PML bodies are not altered in p150CAF-1-depleted ES cells. Immunodetection of PML (red) in ES cells transfected with control (cont) or p150CAF-1 siRNA. The right-hand image shows the merge between PML fluorescence and DAPI-stained DNA in blue.</p></div

Alteration of Epigenetic Marking at Pericentric Heterochromatin in p150CAF-1-Depleted Cells

<div><p>(A) p150CAF-1 depletion leads to reduced H4K20me3 and H3K9me3 at pericentric heterochromatin. Enrichment of histone marks at major satellite repeats was determined by ChIP from control (cont) and p150CAF-1 (p150) siRNA-expressing ES cells. DNA prepared from the input and the antibody-bound fraction were run onto an agarose gel and analyzed by Southern blot with the pSAT major satellite repeat probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b047" target="_blank">47</a>].</p><p>(B) Hybridization signals were quantified using an Instant Imager. After autoradiography, the membrane was stripped and rehybridized with a minor satellite probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b049" target="_blank">49</a>]. After quantification, the membrane was stripped and rehybridized with an IAP LTR probe [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020181#pgen-0020181-b050" target="_blank">50</a>]. Results are presented as the amount of DNA immunoprecipitated from p150CAF-1-depleted ES cells divided by the DNA obtained from control cells. The figure shows the mean value and standard deviation of three independent ChIP experiments.</p><p>(C and D) H3K9me3 and H4K20me3 fluorescence patterns are severely altered in p150CAF-1-depleted ES cells. Immunodetection of H3K9me3 (C, green), H4K20me3 (D, green), and HP1α (red) in control and p150CAF-1 siRNA-expressing ES cells. Merging of HP1α with H3K9me3 (C) and H4K20me3 (D) is shown in yellow. Scale bars represent 10 μm.</p></div