Nuclear Function

Why Study Chromatin?

Conventional wisdom traditionally held that all heredity was specified by the genetic information encoded in the sequence of DNA base pairs that comprise the genome and acquired changes in cell behavior were due to changes in that sequence. While mutations in the coding or regulatory regions of genes are indeed responsible for much of what is of interest to biomedical researchers today, it is clearly not the entire story. The discovery of a second layer of hereditary information, aside from DNA sequence, has given rise to the concept of “epigenetics.” The first described example of epigenetic phenomena related specifically to DNA was hypermethylation that can result in the repression of gene expression. Today, aberrant DNA hypermethylation is known to play a role in a variety of disease states. The most notable example is cancer, which can result when tumor suppressor genes are subject to inappropriate epigenetic inactivation. However, the study of transcriptional co-activators and co-repressors (and their associated enzymatic activities) has led to the proposal that a novel mechanism exists for storing information and regulating genomic function. This hypothesis has been widely referred to as the “histone code,” as this information lies within the variety of post-translational modifications of the histone proteins that organize genomic DNA.

Crystal Structure of the Nucleosome
Crystal structure of the nucleosome, the basic subunit of chromatin. DNA appears in light blue and histones are depicted as colored helices. Post-translational modifications of the histones are represented by black "lollipops". Figure courtesy of Dr. Karolin Luger, Colorado State University.

Within the eukaryotic nucleus, DNA is organized by its incorporation into chromatin. The basic subunit of chromatin is the nucleosome, which is composed of DNA coiled around an octamer of histone proteins, two molecules each of histone H2A, H2B, H3 and H4. Histone H1 associates with chromatin outside the nucleosome and regulates higher order chromatin structure. Each of the histone proteins has an evolutionarily conserved aminoterminal ‘tail’ that protrudes from the nucleosome. This tail is the target of numerous diverse signaling pathways, resulting in the addition of many post-translational modifications. These modifications include phosphorylation, acetylation, methylation, ADP-ribosylation and mono-ubiquitination. Many important new modifications within the structured core and the carboxy-terminal tail regions of histones are also being identified. It is becoming increasingly clear that these modifications represent crucial regulatory events that govern the accessibility and function of the genome

Particular histone modifications, notably acetylation and methylation, have been shown to create potential docking sites on histone tails for proteins with bromodomains or chromodomains (respectively). These binding proteins subsequently regulate chromatin structure and activity. Acetylation and methylation both occur on the epsilon amino group of lysines, but the two modifications appear to be mutually exclusive. In addition to lysines, arginine residues are also post-translationally methylated. The sites of phosphorylation are frequently adjacent to lysine residues that are acetylated or methylated. Phosphorylation may function to disrupt protein complexes recruited to chromatin during cellular processes such as mitosis or apoptosis. In short, virtually every response a cell makes that involves the nucleus will involve changes in chromatin activity by influencing histone modifications and thus altering the histone code.

Diagram of the S. pombe cenH locus, with nearby mating type genes (mat.) and chromatin boundary elements (IR-L and IR-R).

Regions of transcriptionally active euchromatin (blue bar) and inactive heterochromatin (red bar) are indicated below the schematic of the locus. Shown graphically at the bottom of the figure, histone H3 lysine 4 methylation (blue) and H3 lysine 9 mehtylation (red) are associated with discrete regions of this locus, as determined by chromatin immunoprecipitation. Lysine 4 methylation is limited to the active regions of euchromatin, whereas high levels of lysine 9 methylation are detected only in transcriptionally silent heterochromatin. Figure courtesy of Shiv Grewal, NCI.

The study of the histone code has significant potential to enhance current drug development strategies. Inhibitors of histone deacetylases (HDACs) are already in various clinical trials, as DNA methylation is correlated with histone deacetylation and the repression of transcription. Thus, treating cancer cells with HDAC inhibitors can reactivate the expression of silent tumor suppressors stimulating cancer cells to differentiate. The sites deposited by histone modifying enzymes such as methyltansferase and acetyltransferases can be linked to specific diseases (by Chromatin IP), and the enzymes responsible become targets for the treatment of the disease. Every biological process likely has a particular histone modification signature. In principle there could be a chromatin-based drug target for every disease, although much research into the basic biology of chromatin regulation remains to be done. Furthermore, there may be unique surrogate markers in the chromatin modifications that are hallmarks of each biological process.

	Transcriptional Activation	Transcriptional Repression
Acetylation	Increased	Decreased
Lysine methylation	Histone H3 K4	Histone H3 K9, K27, K79
Arginine methylation	HIstone H3 R2 R17, R26 Histone H4 R3	Decreased

Histone H3 Serine 10 Phosphorylation Asynchronous HeLa cells stained with anti-phospho H3 (Ser10) in red and anti-beta tubulin (cat. #05-661) in green. Serine 10 phosphorylation of H3 is almost exclusively observed on condensing mitotic chromosomes (arrows).

Histone H3 Methylation and X-inactivation
Co-localization of Xist RNA and trimethyl histone H3 (Lys 27) on the inactive female X chromosome. Images kindly provided by Drs. Yi Zhang and Barbara Panning (Plath et al., Science 300: 131–135).

H2A.X Tail
Alignment of C-terminal tail regions for histones H2A and H2A.X (human). Non-conserved changes are in red, and sites of phosphorylation are in bold. The site of phosphorylation of H2A.X at serine 139 is indicated by the Greek letter ã.

Antibodies to Modified Histones as "Biomarkers"

It is likely that every biological process has a particular histone modification signature associated with it, and conversely, certain histone modifications are indicative of specific biological processes. Anyone studying these processes can use antibodies that recognize the individual histone modifications as surrogate markers, in many cases simplifying the assays involved.

Transcription

The earliest connection between histone modifications and biological activity came from the observed correlation between histone acetylation and transcription. Increases in acetylation were coupled to higher rates of transcription, and lowered acetylation corresponded with decreased transcription. The explanation for this came many years later, when it was determined that transcriptional activating complexes contain histone acetyltransferases which acetylate histones present at a particular locus, whereas repressor complexes recruit histone deacetylases that remove acetyl groups.

The simple correlation between histone acetylation and gene expression was complicated by the discovery that histone methylation is associated with both increases and decreases in transcription. In addition to increased histone acetylation, the recruitment of proteins that activate transcription also correlates with the methylation of specific lysine and arginine residues. These modifications are catalyzed by enzymes termed histone methyltransferases (HMTs). The net result of these modifications is the creation of a more open chromatin conformation (euchromatin) that facilitates gene expression.

The process of gene activation is at odds with the repressor mediated recruitment of HDACs and a set of HMTs that methylate different lysine residues. Consequently, a more compact chromatin environment (heterochromatin) results, one that is refractory to gene expression. Thus, certain sites of histone methylation are involved in the activation of gene expression, namely lysine 4 of histone H3. A different class of methylation sites on histones is correlated with repression, such as lysines 9, 27 and 79 on histone H3. Using antibodies to specific histone modifications, in concert with the ChIP technique, the transcriptional competence of a region of the genome can be determined and correlated with other phenomena.

Immunofluoresence of HeLa cells using anti-phospho-Histone H2A.X (Ser139), clone JBW301.
Cells were treated with 1 ìg/mL Staurosporine for two hours to induce DNA damage and apoptosis. Left panel, FITC anti-phospho-Histone H2A.X (Ser139). Middle panel, DNA stained with DAPI. Right panel, phase contrast image.

Mitosis

Other biological processes are significantly associated with specific histone modifications, mitosis being a prime example of such a process. During mitosis, several histone phosphorylation events occur, many of which are catalyzed by the Aurora B kinase. These include phosphorylation of histone H3 on serine 10, threonine 11, serine 28, and the phosphorylation of serine 7 on the histone H3 variant CENP-A. The modifications, although specific for mitosis, are detectable at different stages within mitosis, indicating that they likely serve discrete functions during nuclear division. In addition to phosphorylation, methylation of lysine 20 on histone H4 also appears to increase during mitosis.

DNA Damage and Apoptosis

Other examples of cellular processes that involve distinct histone modifications include DNA damage and apoptosis. One of the principal responses to DNA damage resulting in double-strand DNA breaks is the activation of the ATM-initiated signaling cascade to arrest cell division until repairs can be made. One of the major substrates of the kinases (which include CHK1 and CHK2) in this cascade is the histone variant H2A.X. Histone variants are proteins similar in sequence to the canonical histones, but are encoded by discrete genes, exhibit unique expression patterns and serve novel functions within the cell. Phosphorylation occurs at residues in the unique carboxy-terminal tail of H2A.X and is important for recruitment and maintenance of the DNA repair machinery to the site of the break. Interestingly, yeast have only this variant type of H2A, suggesting that it represents the primordial H2A isoform. When the DNA damage is severe enough, the cell will undergo apoptosis and thus H2A.X phosphorylation can be used as a marker for this process. Since this modification occurs very early in apoptosis, long before many of the morphological changes are detectable, it is one of the earliest indicators of apoptosis available.

During the latter stages of apoptosis, Caspase 3 cleaves and activates the protein kinase Mst1, which phosphorylates histone H2B on serine 14. This modification is detectable during the chromatin condensation step that precedes genome fragmentation and is an excellent marker for cells that have committed to the apoptotic pathway. Phosphorylation of serine 32 on H2B has also been reported to increase during apoptosis.

Conclusions and Perspectives

The rapid advancement in our understanding of how chromatin is regulated by histone modifications, and the development of the histone code hypothesis, has raised more possibilities for further discovery and drug design than there are resources available to undertake the effort. Truly this represents a new frontier in biological sciences, where both genetic and epigenetic information will be considered equally important. To facilitate your understanding of the rapidly evolving field of chromatin biology, please visit www.histone.com. This unique resource provides information to the research community regarding histone modifications, the enzymes that deposit these marks, and insights into their biological relevance.

H2B Phosphorylation During Apoptosis
Detection of histone H2B phosphorylated at serine 14 with anti-phospho-histone H2B (Ser14) by immunofluorescence in HL-60 cells stimulated to undergo apoptosis by treatment with VP-16. Top panel, anti-phospho-histone H2B (Ser14) (Millipore cat. no. 07-191) staining. Bottom panel, DAPI staining. Note the distinctive morphology of the nuclear DNA as it breaks down, concommitant with the detection of H2B serine 14 phosphorylation (arrows). Courtesy David Allis, The Rockefeller University.

Mitosis Markers

Interphase G2/M Early Mitosis Late Mitosis

H3 S10 Phos +/– + +++ ++++

H3 S28 Phos – – ++ +++

CENP-A Ser 7 Phos – – +++ +

H4 K20 Me + ++ +++ +++

	Interphase	G2/M	Early Mitosis	Late Mitosis
H3 S10 Phos	+/–	+	+++	++++
H3 S28 Phos	–	–	++	+++
CENP-A Ser 7 Phos	–	–	+++	+
H4 K20 Me	+	++	+++	+++

Several histone modifications appear or are enriched during mitosis. The modifications are listed down the left side of the table, and the stage in cell division with which they are correlated appears across the top. The relative level of staining for each modification is indicated by “minus” or “plus” signs in the table.

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