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.     

Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation.

The retinoblastoma protein (pRb) inhibits progression through the cell cycle. Although pRb is phosphorylated when G1 cyclin-dependent kinases (Cdks) are active, the mechanisms underlying pRb regulation are unknown. In vitro phosphorylation by cyclin D1/Cdk4 leads to inactivation of pRb in a microinjection-based in vivo cell cycle assay. In contrast, phosphorylation of pRb by Cdk2 or Cdk3 in complexes with A- or E-type cyclins is not sufficient to inactivate pRb function in this assay, despite extensive phosphorylation and conversion to a slowly migrating "hyperphosphorylated form." The differential effects of phosphorylation on pRb function coincide with modification of distinct sets of sites. Serine 795 is phosphorylated efficiently by Cdk4, even in the absence of an intact LXCXE motif in cyclin D, but not by Cdk2 or Cdk3. Mutation of serine 795 to alanine prevents pRb inactivation by Cdk4 phosphorylation in the microinjection assay. This study identifies a residue whose phosphorylation is critical for inactivation of pRb-mediated growth suppression, and it indicates that hyperphosphorylation and inactivation of pRb are not necessarily synonymous.     

A new regulator of the cell cycle

The ability of eukaryotes to alter chromatin structure and function is modulated, in part, by histone-modifying enzymes and the post-translational modifications they create. One of these enzymes, PR-Set7/Set8/KMT5a, is the sole histone methyltransferase responsible for the monomethylation of histone H4 lysine 20 (H4K20me1) in higher eukaryotes. Both PR-Set7 and H4K20me1 were previously found to be tightly cell cycle regulated suggesting that they play an important, although unknown, role in cell cycle progression. Several recent reports reveal that PR-Set7 abundance is dynamically regulated during different cell cycle phases by distinct enzymes including cdk1/cyclinB, Cdc14, SCF^Skp2, CRL4^cdt2 and APC^cdh1. Importantly, these reports demonstrate that inappropriate levels of PR-Set7 result in profound cell cycle defects including the inability to initiate S phase, the re-replication of DNA and the improper timing of mitotic progression. Here, we summarize the significance of these new findings, raise some important questions that require further investigation and explore several possibilities of how PR-Set7 and methylated H4K20 may likely function as novel regulators of the cell cycle.     

A new regulator of the cell cycle: The PR-Set7 histone methyltransferase

The ability of eukaryotes to alter chromatin structure and function is modulated, in part, by histone-modifying enzymes and the post-translational modifications they create. One of these enzymes, PR-Set7/Set8/KMT5a, is the sole histone methyltransferase responsible for the monomethylation of histone H4 lysine 20 (H4K20me1) in higher eukaryotes. Both PR-Set7 and H4K20me1 were previously found to be tightly cell cycle regulated suggesting that they play an important, although unknown, role in cell cycle progression. Several recent reports reveal that PR-Set7 abundance is dynamically regulated during different cell cycle phases by distinct enzymes including cdk1/cyclinB, Cdc14, SCF^Skp2, CRL4^cdt2 and APC^cdh1. Importantly, these reports demonstrate that inappropriate levels of PR-Set7 result in profound cell cycle defects including the inability to initiate S phase, the re-replication of DNA and the improper timing of mitotic progression. Here, we summarize the significance of these new findings, raise some important questions that require further investigation and explore several possibilities of how PR-Set7 and methylated H4K20 may likely function as novel regulators of the cell cycle.Key words: PR-Set7, Set8, histone H4, methylation, ubiquitination, epigenetic, chromatin, SCF^Skp2, CRL4^cdt2, APC^cdh1, cdk1/cyclinB, Cdc14     

芽殖酵母和裂殖酵母中异染色质形成机制

芽殖酵母(Saccharomyces cerevisiae)和裂殖酵母(Schizosaccharomyces pombe)是用来研究异染色质形成、细胞周期、DNA复制等重要细胞功能的理想单细胞真核生物.本文主要介绍这2种酵母中异染色质形成的机制.异染色质是一种抑制基因转录和DNA重组的特殊染色质结构.尽管在芽殖酵母和裂殖酵母中异染色质形成都需要组蛋白修饰,但异染色质建立的机制不同.在芽殖酵母中参与异染色质形成的主要蛋白是Sir1-4蛋白（其中Sir2为组蛋白H3去乙酰化酶）,而组蛋白H3赖氨酸9甲基化酶Clr4和异染色质蛋白Swi6在裂殖酵母异染色质形成中起关键的作用.在这两个酵母中,参与异染色质形成的组蛋白修饰蛋白由DNA结合蛋白招募到异染色质.此外,裂殖酵母也利用RNA干扰系统招募组蛋白修饰蛋白.     

Dynamic Regulation of Effector Protein Binding to Histone Modifications: The Biology of HP1 Switching

Post-translational modifications of histone proteins, the basic building blocks around which eukaryotic DNA is organized, are crucially involved in the regulation of genome activity as they control chromatin structure and dynamics. The recruitment of specific binding proteins that recognize and interact with particular histone modifications is thought to constitute a fundamental mechanism by which histone marks mediate biological function. For instance, tri-methylation of histone H3 lysine 9 (H3K9me3) is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging, and heterochromatin formation. Until now, little was known about the regulation of effector-histone mark interactions, and in particular, of the binding of HP1 to H3K9me3. Recently, we and others presented evidence that a "binary methylation-phosphorylation switch" mechanism controls the dynamic release of HP1 from H3K9me3 during the cell cycle: phosphorylation of histone H3 serine 10 (H3S10ph) occurs at the onset of mitosis, interferes with HP1-H3K9me3 interaction, and therefore, ejects HP1 from its binding site. Here, we discuss the biological function of HP1 release from chromatin during mitosis, consider implications why the cell controls HP1 binding by such a methylation-phosphorylation switching mechanism, and reflect on other cellular pathways where binary switching of HP1 might occur.     

Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage

Histone lysine methylation is a key regulator of gene expression and heterochromatin function, but little is known as to how this modification impinges on other chromatin activities. Here we demonstrate that a previously uncharacterized SET domain protein, Set9, is responsible for H4-K20 methylation in the fission yeast Schizosaccharomyces pombe. Surprisingly, H4-K20 methylation does not have any apparent role in the regulation of gene expression or heterochromatin function. Rather, we find the modification has a role in DNA damage response. Loss of Set9 activity or mutation of H4-K20 markedly impairs cell survival after genotoxic challenge and compromises the ability of cells to maintain checkpoint mediated cell cycle arrest. Genetic experiments link Set9 to Crb2, a homolog of the mammalian checkpoint protein 53BP1, and the enzyme is required for Crb2 localization to sites of DNA damage. These results argue that H4-K20 methylation functions as a "histone mark" required for the recruitment of the checkpoint protein Crb2.     

Interplay between histone deacetylase SIR-2, linker histone H1 and histone methyltransferases in heterochromatin formation

Maintenance of intact heterochromatin structure through epigenetic mechanisms is essential for cell survival. Defects in heterochromatin formation caused by loss of chromatin-modifying enzymes lead to genomic instability and cellular senescence. The NAD+-dependent histone deacetylase SIR-2 and the H1 linker histone are intriguing chromatin elements that are connected to chromatin regulation and cell viability in the single cellular eukaryotic organism yeast. However, it remains an open question how SIR-2 and H1 mediate heterochromatin formation in simple multi-cellular organisms such as C. elegans and in even more complex organisms such as mammals. Recently we have identified SIR-2.1 and the H1 histone subtype, HIS-24 as factors involved in heterochromatin regulation at subtelomeric regions in C. elegans. In addition we show that SIR-2.1, HIS-24, and MES-2, a ortholog to Enhancer of zeste E(Z) are functionally related in heterochromatin formation contributing to fertility and embryogenesis. Here we discuss the interplay between SIR-2, H1 histone and histone methyltransferases in modulation of chromatin structure in further detail.     

Control of gene expression and assembly of chromosomal subdomains by chromatin regulators with antagonistic functions

Epigenetic regulation of higher-order chromatin structure controls gene expression and the assembly of chromosomal domains during cell division, differentiation, and development. The proposed “histone code” integrates a complex system of histone modifications and chromosomal proteins that establish and maintain distinctive types of chromatin, such as euchromatin, heterochromatin, and centromeric (CEN) chromatin. The reversible nature of histone acetylation, phosphorylation, and (most recently discovered) methylation are mechanisms for controlling gene expression and partitioning the genome into functional domains. Many different regions of the genome contain similar epigenetic marks (histone modifications), raising the question as to how they are independently specified and regulated. In this review, we will focus on several recent discoveries in chromatin and chromosome biology: (1) identification of long-elusive histone “de-methylating” enzymes that affect chromatin structure, and (2) assembly and maintenance of chromatin domains, specifically heterochromatin and euchromatin, through a dynamic equilibrium of modifying enzymes, histone modifications, and histone variants identified biochemically and genetically. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK     

Molecular determinants for the complex formation between the retinoblastoma protein and LXCXE sequences

The retinoblastoma tumor suppressor protein (pRb) is a key negative regulator of cell proliferation that is frequently disregulated in human cancer. Many viral oncoproteins (for example, HPV E7 and E1A) are known to bind to the pRb pocket domain via a LXCXE binding motif. There are also some 20 cellular proteins that contain a LXCXE motif and have been reported to associate with the pocket domain of pRb. Using NMR spectroscopy and isothermal calorimetry titration, we show that LXCXE peptides of viral oncoproteins bind strongly to the pocket domain of pRb. Additionally, we show that LXCXE-like peptides of HDAC1 bind to the same site on pRb with a weak (micromolar) and transient association. Systematic substitution of residues other than conserved Leu, Cys, and Glu show that the residues flanking the LXCXE are important for the binding, whereas positively charged amino acids in the XLXCXEXXX sequence significantly weaken the interaction.     

Role of the RB1 family in stabilizing histone methylation at constitutive heterochromatin

Here, we show a role for the RB1 family proteins in directing full heterochromatin formation. Mouse embryonic fibroblasts that are triply deficient for RB1 (retinoblastoma 1), RBL1 (retinoblastoma-like 1) and RBL2 (retinoblastoma-like 2) - known as TKO cells - show a marked genomic instability, which is coincidental with decreased DNA methylation, increased acetylation of histone H3 and decreased tri-methylation of histone H4 at lysine 20 (H4K20). Chromatin immunoprecipitation showed that H4K20 tri-methylation was specifically decreased at pericentric and telomeric chromatin. These defects are independent of E2F family function. Indeed, we show a direct interaction between the RB1 proteins and the H4K20 tri-methylating enzymes Suv4-20h1 and Suv4-20h2, indicating that the RB1 family has a role in controlling H4K20 tri-methylation by these histone methyltransferases. These observations indicate that the RB1 family is involved in maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin, linking tumour suppression and the epigenetic definition of chromatin.     

The PHD finger of human UHRF1 reveals a new subgroup of unmethylated histone H3 tail readers

The human UHRF1 protein (ubiquitin-like containing PHD and RING finger domains 1) has emerged as a potential cancer target due to its implication in cell cycle regulation, maintenance of DNA methylation after replication and heterochromatin formation. UHRF1 functions as an adaptor protein that binds to histones and recruits histone modifying enzymes, like HDAC1 or G9a, which exert their action on chromatin. In this work, we show the binding specificity of the PHD finger of human UHRF1 (huUHRF1-PHD) towards unmodified histone H3 N-terminal tail using native gel electrophoresis and isothermal titration calorimetry. We report the molecular basis of this interaction by determining the crystal structure of huUHRF1-PHD in complex with the histone H3 N-terminal tail. The structure reveals a new mode of histone recognition involving an extra conserved zinc finger preceding the conventional PHD finger region. This additional zinc finger forms part of a large surface cavity that accommodates the side chain of the histone H3 lysine K4 (H3K4) regardless of its methylation state. Mutation of Q330, which specifically interacts with H3K4, to alanine has no effect on the binding, suggesting a loose interaction between huUHRF1-PHD and H3K4. On the other hand, the recognition appears to rely on histone H3R2, which fits snugly into a groove on the protein and makes tight interactions with the conserved aspartates D334 and D337. Indeed, a mutation of the former aspartate disrupts the formation of the complex, while mutating the latter decreases the binding affinity nine-fold.