Akash Gunjan

Dynamic binding of histone H1 to chromatin in living cells

The linker histone H1 is believed to be involved in chromatin organization by stabilizing higher-order chromatin structure. Histone H1 is generally viewed as a repressor of transcription as it prevents the access of transcription factors and chromatin remodelling complexes to DNA. Determining the binding properties of histone H1 to chromatin in vivo is central to understanding how it exerts these functions. We have used photobleaching techniques to measure the dynamic binding of histone H1–GFP to unperturbed chromatin in living cells. Here we show that almost the entire population of H1–GFP is bound to chromatin at any one time; however, H1–GFP is exchanged continuously between chromatin regions. The residence time of H1–GFP on chromatin between exchange events is several minutes in both euchromatin and heterochromatin. In addition to the mobile fraction, we detected a kinetically distinct, less mobile fraction. After hyperacetylation of core histones, the residence time of H1–GFP is reduced, suggesting a higher rate of exchange upon chromatin remodelling. These results support a model in which linker histones bind dynamically to chromatin in a stop-and-go mode.

A Rad53 Kinase-Dependent Surveillance Mechanism that Regulates Histone Protein Levels in S. cerevisiae

Rad53 and Mec1 are protein kinases required for DNA replication and recovery from DNA damage in Saccharomyces cerevisiae. Here, we show that rad53, but not mec1 mutants, are extremely sensitive to histone overexpression, as Rad53 is required for degradation of excess histones. Consequently, excess histones accumulate in rad53 mutants, resulting in slow growth, DNA damage sensitivity, and chromosome loss phenotypes that are significantly suppressed by a reduction in histone gene dosage. Rad53 monitors excess histones by associating with them in a dynamic complex that is modulated by its kinase activity. Our results argue that Rad53 contributes to genome stability independently of Mec1 by preventing the damaging effects of excess histones both during normal cell cycle progression and in response to DNA damage.

Histone levels are regulated by phosphorylation and ubiquitylation dependent proteolysis

Histone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and seem to undergo modifications such as tyrosine phosphorylation and polyubiquitylation, before their proteolysis by the proteasome. We have identified the Tyr 99 residue of histone H3 as being critical for the efficient ubiquitylation and degradation of this histone. We have also identified the ubiquitin conjugating enzymes (E2) Ubc4 and Ubc5, as well as the ubiquitin ligase (E3) Tom1 (temperature dependent organization in mitotic nucleus 1), as enzymes involved in the ubiquitylation of excess histones. Regulated histone proteolysis has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA.

Excess histone levels mediate cytotoxicity via multiple mechanisms.

The accumulation of excess histone proteins in cells has deleterious consequences such as genomic instability in the form of excessive chromosome loss, enhanced sensitivity to DNA damaging agents and cytotoxicity. Hence, the synthesis of histone proteins is tightly regulated at multiple steps and transcriptional as well as posttranscriptional regulation of histone proteins is well established. Additionally, we have recently demonstrated that histone protein levels are regulated posttranslationally by the DNA damage checkpoint kinase Rad53 and ubiquitin-proteasome dependent proteolysis in the budding yeast. However, the underlying mechanism/s via which excess histones exert their deleterious effects in vivo are not clear. Here we have investigated the mechanistic basis for the deleterious effects of excess histones in budding yeast. We find that the presence of excess histones saturates certain histone modifying enzymes, potentially interfering with their activities. Additionally, excess histones appear to bind non-specifically to DNA as well as RNA, which can adversely affect their metabolism. Microarray analysis revealed that upon overexpression of histone gene pairs, about 240 genes were either up- or downregulated by 2-fold or more. Overall, we present evidence that excess histones are likely to mediate their cytotoxic effects via multiple mechanisms that are primarily dependent on inappropriate electrostatic interactions between the positively charged histones and diverse negatively charged molecules in the cell. Our findings help explain the basis for the existence of multiple distinct mechanisms that contribute to the tight control of histone protein levels in cells and highlight their importance in maintaining genomic stability and cell viability.

Effects of H1 Histone Variant Overexpression on Chromatin Structure

The importance of histone H1 heterogeneity and total H1 stoichiometry in chromatin has been enigmatic. Here we report a detailed characterization of the chromatin structure of cells overexpressing either H10 or H1c. Nucleosome spacing was found to change during cell cycle progression, and overexpression of either variant in exponentially growing cells results in a 15-base pair increase in nucleosome repeat length. H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication. Overexpression of H10 and, to a lesser extent, H1c results in a decreased rate of digestion of chromatin by micrococcal nuclease. Using green fluorescent protein-tagged H1 variants, we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H10 variant. Overexpression of H10 results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels. Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H10 protein could independently give rise to this unique mononucleosome species. These results in part explain the differential effects of H10 and H1c in regulating chromatin structure and function.

FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication.

Novel E3 ubiquitin ligases that regulate histone protein levels in the budding yeast Saccharomyces cerevisiae.

Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus) domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene) finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs) YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases) respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3 ubiquitin ligases to ensure excess histone degradation and thus contribute to the maintenance of genomic stability.

Histone tyrosine phosphorylation comes of age

Histones were discovered over a century ago and have since been found to be the most extensively posttranslationally modified proteins, although tyrosine phosphorylation of histones had remained elusive until recently. The year 2009 proved to be a landmark year for histone tyrosine (Y) phosphorylation as five research groups independently discovered this modification. Three groups describe phosphorylation of Y142 in the variant histone H2A.X, where it may be involved in the cellular decision making process to either undergo DNA repair or apoptosis in response to DNA damage. Further, one group suggests that phosphorylation of histone H3 on Y99 is crucial for its regulated proteolysis in yeast, while another found that Y41 phosphorylation modulates chromatin architecture and oncogenesis in mammalian cells. These pioneering studies provide the initial conceptual framework for further analyses of the diverse roles of tyrosine phosphorylation on different histones, with far reaching implications for human health and disease. Erratum to: Singh R.K. and Gunjan A. Histone tyrosine phosphorylation comes of age. Epigenetics 2011; 6:153-60.

Histone dosage regulates DNA damage sensitivity in a checkpoint-independent manner by the homologous recombination pathway

In eukaryotes, multiple genes encode histone proteins that package genomic deoxyribonucleic acid (DNA) and regulate its accessibility. Because of their positive charge, ‘free’ (non-chromatin associated) histones can bind non-specifically to the negatively charged DNA and affect its metabolism, including DNA repair. We have investigated the effect of altering histone dosage on DNA repair in budding yeast. An increase in histone gene dosage resulted in enhanced DNA damage sensitivity, whereas deletion of a H3–H4 gene pair resulted in reduced levels of free H3 and H4 concomitant with resistance to DNA damaging agents, even in mutants defective in the DNA damage checkpoint. Studies involving the repair of a HO endonuclease-mediated DNA double-strand break (DSB) at the MAT locus show enhanced repair efficiency by the homologous recombination (HR) pathway on a reduction in histone dosage. Cells with reduced histone dosage experience greater histone loss around a DSB, whereas the recruitment of HR factors is concomitantly enhanced. Further, free histones compete with the HR machinery for binding to DNA and associate with certain HR factors, potentially interfering with HR-mediated repair. Our findings may have important implications for DNA repair, genomic stability, carcinogenesis and aging in human cells that have dozens of histone genes.

Generation and management of excess histones during the cell cycle.

Histones are essential proteins that package the DNA in all eukaryotes into chromosomes. However, histones can accumulate upon a decrease in DNA synthesis that occurs at the end of S-phase or following replication arrest. These positively charged histones can associate non-specifically with the negatively charged DNA and other cellular biomolecules, impairing their normal function. Hence, cells have evolved numerous strategies to limit the generation of excess histones and prevent deleterious effects due to their accumulation. Such strategies for histone regulation are discussed here, with particular emphasis on recent studies that implicate the DNA damage checkpoint kinases in the regulation of histone levels, especially in response to replication inhibition. We have also focused upon the recently discovered regulatory mechanism involving histone proteolysis in the budding yeast. Additionally, we speculate that cells may possess a surveillance mechanism for sensing histone levels, particularly in the G1 and S-phases of the cell cycle. Proper regulation of histone levels has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA.