Integration of molecular genetics and proteomics with cell metabolism: how to proceed; how not to proceed!

There now exists a resurgence of interest in the role of intermediary metabolism in medicine; especially in relation to medical disorders. Coupled with this is the contemporary focus on molecular biology, genetics and proteomics and their integration into studies of regulation and alterations in cellular metabolism in health and disease. This is a marriage that has vast potential for elucidation of the factors and conditions that are involved in cellular metabolic and functional changes, which heretofore could not be addressed by the earlier generations of biochemists who established the major pathways of intermediary metabolism. The achievement of this present potential requires the appropriate application and interpretation of genetic and proteomic studies relating to cell metabolism and cell function. This requires knowledge and understanding of the principles, relationships, and methodology, such as biochemistry and enzymology, which are involved in the elucidation of cellular regulatory enzymes and metabolic pathways. Unfortunately, many and possibly most contemporary molecular biologists are not adequately trained and knowledgeable in these areas of cell metabolism. This has resulted in much too common inappropriate application and misinformation from genetic/proteomic studies of cell metabolism and function. This presentation describes important relationships of cellular intermediary metabolism, and provides examples of the appropriate and inappropriate application of genetics and proteomics. It calls for the inclusion of biochemistry, enzymology, cell metabolism and cell physiology in the graduate and postgraduate training of molecular biology and other biomedical researchers.     

Histone phosphorylation during sea urchin development

Studies on histone phosphorylation during transitions in chromatin structure occurringin vivoduring spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extractsin vitroand evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structurein vivoand provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.     

Histone phosphorylation during liver regeneration.

The pattern of histone phosphorylation at acid-stable, alkali-labile sites has been examined throughout the early stages of liver regeneration, namely at times of “gene activation”. Among the histones, only H1 shows an increase in phosphorylation. This increase initiates near the end of the period of chromatin template activation. Thus, there is no obvious temporal correlation between increased histone phosphorylation and increased RNA synthesis. The relative levels of phosphorylation of the various histones and the change in H1 phosphorylation observed in the liver system closely parallel the patterns exhibited by cultured animal cells during the G1 and S phases of the cycle as described by other investigators.     

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 tyrosine phosphorylation comes of age

Histones were discovered over a century ago and have since been found to be the most extensively post-translationally 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.Key words: histones, chromatin, tyrosine phosphorylation, genomic instability, DNA damage, DNA repair, apoptosis, ubiquitylation, proteolysis, cancer     

Histone phosphorylation in late interphase and mitosis

Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the $\text{G}{}_1\text{S}$ boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G₂-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G₁, S, or G₂-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.     

Histone H2A phosphorylation in animal cells: functional considerations

We have sought to determine the role of histone H2A phosphorylation in chromatin by examining the distribution of the phosphorylated and unphosphorylated forms of this core histone within the nuclei of mouse and human cells. At any time, only about 15% of the H2A of whole chromatin is in the phosphorylated form, and its phosphate is rapidly turned over, even in quiescent cells that contain a functional nucleus. The phosphorylations and dephosphorylations are not specifically relate to progress through the cell cycle, nor to DNA synthesis or repair, and they are not selectively nucleolar. Euchromatin is substantially enriched with phosphorylated H2A but is not the exclusive repository of it. Possible roles of this modification of H2A are considered.     

Histone phosphorylation integrates the hepatic glucagon-PKA-CREB gluconeogenesis program in response to fasting

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Histone phosphorylation in explants of mouse mammary glands and tumors

Rates of histone phosphorylation were measured in explants of mammary glands from mouse strains with high and low tumor incidence. Explants of hormone dependent and independent mouse mammary tumors were also investigated. All mouse strains studied showed predominant phosphorylation of H2A histone at serine and threonine residues. No differences in rates of H2A phosphorylation in glands were found between strains having different mammary tumor susceptibility. Hormone-dependent GR mouse mammary tumors also showed high H2A phosphorylation, but in some tumors also H1 and H3 were phosphorylated. Hormone-dependent GR tumors had 2–5 times higher histone phosphorylation at serine and threonine than hormone-independent tumors.     

Histone H2A phosphorylation in DNA double-strand break repair

DNA repair must take place within the context of chromatin, and it is therefore not surprising that many aspects of both chromatin components and proteins that modify chromatin have been implicated in this process. One of the best-characterized chromatin modification events in DNA-damage responses is the phosphorylation of the SQ motif found in histone H2A or the H2AX histone variant in higher eukaryotes. This modification is an early response to the induction of DNA damage, and occurs in a wide range of eukaryotic organisms, suggesting an important conserved function. One function that histone modifications can have is to provide a unique binding site for interacting factors. Here, we review the proteins and protein complexes that have been identified as H2AS129ph (budding yeast) or H2AXS139ph (human) binding partners and discuss the implications of these interactions.     

Histone H4 histidine phosphorylation: kinases, phosphatases, liver regeneration and cancer

Phosphorylation of histone H4 on one or both of its two histidine residues has been known to occur in liver cells for nearly 40?years and has been associated with proliferation of hepatocytes during regeneration of the liver following mechanical damage. More recently, large increases in histone H4 histidine kinase activity have been found to occur associated with proliferation and differentiation of liver progenitor cells following chemical damage that prevents hepatocyte proliferation. In addition, it has been shown this histone H4 histidine kinase activity is elevated nearly 100-fold in human foetal liver and several hundredfold in hepatocellular carcinoma tissue compared with normal adult liver. In the present paper, we review what is currently known about histone H4 histidine phosphorylation, the kinase(s) responsible and the phosphatases capable of catalysing its dephosphorylation, and briefly summarize the techniques used to detect and measure the histidine phosphorylation of histone H4 and the corresponding kinase activity.