CpG-binding protein (CXXC finger protein 1) is a component of the mammalian Set1 histone H3-Lys4 methyltransferase complex, the analogue of the yeast Set1/COMPASS complex

CpG-binding protein (CXXC finger protein 1 (CFP1)) binds to DNA containing unmethylated CpG motifs and is required for mammalian embryogenesis, normal cytosine methylation, and cellular differentiation. Studies were performed to identify proteins that interact with CFP1 to gain insight into the molecular function of this protein. Immunoprecipitation and mass spectrometry reveal that human CFP1 associates with a approximately 450-kDa complex that contains the mammalian homologues of six of the seven components of the Set1/COMPASS complex, the sole histone H3-Lys4 methyltransferase in yeast. In vitro assays demonstrate that the human Set1/CFP1 complex is a histone methyltransferase that produces mono-, di-, and trimethylated histone H3 at Lys4. Confocal microscopy reveals that CFP1 and Set1 co-localize to nuclear speckles associated with euchromatin. A Set1 complex of reduced mass persists in murine embryonic stem cells lacking CFP1. These cells carry elevated levels of methylated histone H3-Lys4 and reduced levels of methylated histone H3-Lys9. Together with the previous finding of reduced levels of cytosine methylation, these data indicate that cells lacking CFP1 contain reduced levels of heterochromatin. Furthermore, ES cells lacking CFP1 exhibit a 4-fold excess of histone H3-Lys4 methylation following induction of differentiation, indicating that CFP1 restricts the activity of the Set1 histone methyltransferase complex. These results reveal a mammalian counterpart to the yeast Set1/COMPASS complex. The presence of CFP1 in this complex implicates this protein as a critical epigenetic regulator of histone modification in addition to cytosine methylation and reveals one mechanism by which this protein intersects with the epigenetic machinery.     

DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction

DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.     

Epigenetic regulation of neonatal cardiomyocytes differentiation

The relationship between DNA methylation, histone modifications and terminal differentiation in cardiomyocytes was investigated in this study. The upregulation of methylation-related proteins, including DNA methyltransferase (DNMT) 1, methyl-CpG binding domain proteins 1, 2 and 3, and the increase in global methylation during rat neonatal heart development were observed. Moreover, an increase in DNA synthesis and a delay in differentiation were found in 5-azacytidine (5-azaC)-treated cardiomyocytes. Increase in acetylation of H3-K9, H4-K5, H4-K8 and methylation of H3-K4 suggested a more accessible chromatin structure in 5-azaC-treated cells. Furthermore, methyl-CpG-binding protein 2 was found to be upregulated in differentiated cardiomyocytes. Overexpression of this protein resulted in an increase of global methylation levels. Therefore, we suggest that a hypermethylated genome and a more compact chromatin structure are formed during terminal differentiation of cardiomyocytes.     

KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

Background

Pluripotency of embryonic stem (ES) cells is controlled in part by chromatin-modifying factors that regulate histone H3 lysine 4 (H3K4) methylation. However, it remains unclear how H3K4 demethylation contributes to ES cell function.

Results

Here, we show that KDM5B, which demethylates lysine 4 of histone H3, co-localizes with H3K4me3 near promoters and enhancers of active genes in ES cells; its depletion leads to spreading of H3K4 methylation into gene bodies and enhancer shores, indicating that KDM5B functions to focus H3K4 methylation at promoters and enhancers. Spreading of H3K4 methylation to gene bodies and enhancer shores is linked to defects in gene expression programs and enhancer activity, respectively, during self-renewal and differentiation of KDM5B-depleted ES cells. KDM5B critically regulates H3K4 methylation at bivalent genes during differentiation in the absence of LIF or Oct4. We also show that KDM5B and LSD1, another H3K4 demethylase, co-regulate H3K4 methylation at active promoters but they retain distinct roles in demethylating gene body regions and bivalent genes.

Conclusions

Our results provide global and functional insight into the role of KDM5B in regulating H3K4 methylation marks near promoters, gene bodies, and enhancers in ES cells and during differentiation.     

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei

Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly casually involved in the formation of cell type specific heterochromatin compartments, composed of (peri)centromeric regions and chromosomal subregions from neighboring chromosomes territories, which contain silent genes.     

Fractionated low-dose radiation exposure leads to accumulation of DNA damage and profound alterations in DNA and histone methylation in the murine thymus

Thymus, an important component of hematopoietic tissue, is a well-documented "target" of radiation carcinogenesis. Both acute and fractionated irradiation result in a high risk of leukemia and thymic lymphoma. However, the exact mechanisms underlying radiation-induced predisposition to leukemia and lymphoma are still unknown, and the contributions of genetic and epigenetic mechanisms in particular have yet to be defined. Global DNA hypomethylation is a well-known characteristic of cancer cells. Recent studies have also shown that tumor cells undergo prominent changes in histone methylation, particularly a substantial loss of trimethylation of histone H4-Lys20 and demethylation of genomic DNA. These losses are considered a universal marker of malignant transformation. In the present study, we investigated the effect of low-dose radiation exposure on the accumulation of DNA lesions and alterations of DNA methylation and histone H4-Lys20 trimethylation in the thymus tissue using an in vivo murine model. For the first time, we show that fractionated whole-body application of 0.5 Gy X-ray leads to decrease in histone H4-Lys20 trimethylation in the thymus. The loss of histone H4-Lys20 trimethylation was accompanied by a significant decrease in global DNA methylation as well as the accumulation of DNA damage as monitored by persistence of histone gammaH2AX foci in the thymus tissue of mice exposed to fractionated irradiation. Altered DNA methylation was associated with reduced expression of maintenance (DNMT1) and, to a lesser extent, de novo DNA methyltransferase DNMT3a in exposed animals. Expression of another de novo DNA methyltransferase DNMT3b was decreased only in males. Irradiation also resulted in approximately 20% reduction in the levels of methyl-binding proteins MeCP2 and MBD2. Our results show the involvement of epigenetic alterations in radiation-induced responses in vivo. These changes may play a role in genome destabilization that ultimately leads to cancer.     

The profile of repeat-associated histone lysine methylation states in the mouse epigenome

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints.     

A chromosomal memory triggered by Xist regulates histone methylation in X inactivation

We have elucidated the kinetics of histone methylation during X inactivation using an inducible Xist expression system in mouse embryonic stem (ES) cells. Previous reports showed that the ability of Xist to trigger silencing is restricted to an early window in ES cell differentiation. Here we show that this window is also important for establishing methylation patterns on the potential inactive X chromosome. By immunofluorescence and chromatin immunoprecipitation experiments we show that histone H3 lysine 27 trimethylation (H3K27m3) and H4 lysine 20 monomethylation (H4K20m1) are associated with Xist expression in undifferentiated ES cells and mark the initiation of X inactivation. Both marks depend on Xist RNA localisation but are independent of silencing. Induction of Xist expression after the initiation window leads to a markedly reduced ability to induce H3K27m3, whereas expression before the restrictive time point allows efficient H3K27m3 establishment. Our data show that Xist expression early in ES cell differentiation establishes a chromosomal memory, which is maintained in the absence of silencing. One consequence of this memory is the ability to introduce H3K27m3 efficiently after the restrictive time point on the chromosome that has expressed Xist early. Our results suggest that this silencing-independent chromosomal memory has important implications for the maintenance of X inactivation, where previously self-perpetuating heterochromatin structures were viewed as the principal form of memory.     

A Chromosomal Memory Triggered by Xist Regulates Histone Methylation in X Inactivation

We have elucidated the kinetics of histone methylation during X inactivation using an inducible Xist expression system in mouse embryonic stem (ES) cells. Previous reports showed that the ability of Xist to trigger silencing is restricted to an early window in ES cell differentiation. Here we show that this window is also important for establishing methylation patterns on the potential inactive X chromosome. By immunofluorescence and chromatin immunoprecipitation experiments we show that histone H3 lysine 27 trimethylation (H3K27m3) and H4 lysine 20 monomethylation (H4K20m1) are associated with Xist expression in undifferentiated ES cells and mark the initiation of X inactivation. Both marks depend on Xist RNA localisation but are independent of silencing. Induction of Xist expression after the initiation window leads to a markedly reduced ability to induce H3K27m3, whereas expression before the restrictive time point allows efficient H3K27m3 establishment. Our data show that Xist expression early in ES cell differentiation establishes a chromosomal memory, which is maintained in the absence of silencing. One consequence of this memory is the ability to introduce H3K27m3 efficiently after the restrictive time point on the chromosome that has expressed Xist early. Our results suggest that this silencing-independent chromosomal memory has important implications for the maintenance of X inactivation, where previously self-perpetuating heterochromatin structures were viewed as the principal form of memory.     

Two ubiquitin-conjugating enzymes,Rhp6 and UbcX,regulate heterochromatin silencing in Schizosaccharomyces pombe

Methylation of histone H3 has been linked to the assembly of higher-order chromatin structures. Very recently, several examples, including the Schizosaccharomyces pombe mating-type region, chicken beta-globin locus, and inactive X-chromosome, revealed that H3-Lys9-methyl (Me) is associated with silent chromatin while H3-Lys4-Me is prominent in active chromatin. Surprisingly, it was shown that homologs of Drosophila Su(var)3-9 specifically methylate the Lys9 residue of histone H3. Here, to identify putative enzymes responsible for destabilization of heterochromatin, we screened genes whose overexpressions disrupt silencing at the silent mat3 locus in fission yeast. Interestingly, we identified two genes, rhp6(+) and ubcX(+) (ubiquitin-conjugating enzyme participating in silencing), both of which encode ubiquitin-conjugating enzymes. Their overexpression disrupted silencing at centromeres and telomeres as well as at mat3. Additionally, the overexpression interfered with centromeric function, as confirmed by elevated minichromosome loss and antimicrotubule drug sensitivity. On the contrary, deletion of rhp6(+) or ubcX(+) enhanced silencing at all heterochromatic regions tested, indicating that they are negative regulators of silencing. More importantly, chromatin immunoprecipitation showed that their overexpression alleviated the level of H3-Lys9-Me while enhancing the level of H3-Lys4-Me at the silent regions. On the contrary, their deletions enhanced the level of H3-Lys9-Me while alleviating that of H3-Lys4-Me. Taken together, the data suggest that two ubiquitin-conjugating enzymes, Rhp6 and UbcX, affect methylation of histone H3 at silent chromatin, which then reconfigures silencing.     

Phosphorylation of the linker histone H1 by CDK regulates its binding to HP1alpha

Two key components of mammalian heterochromatin that play a structural role in higher order chromatin organization are the heterochromatin protein 1alpha (HP1alpha) and the linker histone H1. Here, we show that these proteins interact in vivo and in vitro through their hinge and C-terminal domains, respectively. The phosphorylation of H1 by CDK2, which is required for efficient cell cycle progression, disrupts this interaction. We propose that phosphorylation of H1 provides a signal for the disassembly of higher order chromatin structures during interphase, independent of histone H3-lysine 9 (H3-K9) methylation, by reducing the affinity of HP1alpha for heterochromatin.     

Distinct dynamics and distribution of histone methyl-lysine derivatives in mouse development

Histone methylation acts as an epigenetic regulator of chromatin activity through the modification of arginine and lysine residues on histones H3 and H4. In the case of lysine, this includes the formation of mono-, di-, or trimethyl groups, each of which is presumed to represent a distinct functional state at the cellular level. To examine the potential developmental roles of these modifications, we determined the global patterns of lysine methylation involving K9 on histone H3 and K20 on histone H4 in midgestation mouse embryos. For each lysine target site, we observed distinct subnuclear distributions of the mono- and trimethyl versions in 10T1/2 cells that were conserved within primary cultures and within the 3D-tissue architecture of the embryo. Interestingly, three of these modifications, histone H3 trimethyl K9, histone H4 monomethyl K20, and histone H4 trimethyl K20 exhibited marked differences in their distribution within the neuroepithelium. Specifically, both histone H3 trimethyl K9 and H4 monomethyl K20 were elevated in proliferating cells of the neural tube, which in the case of the K9 modification was limited to mitotic cells on the luminal surface. In contrast, histone H4 trimethyl K20 was progressively lost from these medial regions and became enriched in differentiating neurons in the ventrolateral neural tube. The inverse relationship of histone H4 K20 methyl derivatives is even more striking during skeletal and cardiac myogenesis where the accumulation of the trimethyl modification in pericentromeric heterochromatin suggests a role in gene silencing in postmitotic muscle cells. Importantly, our results establish that histone lysine methylation occurs in a highly dynamic manner that is consistent with their function in an epigenetic program for cell division and differentiation.