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MOLECULAR BIOLOGY: ON THE PACKING OF DNA IN NUCLEOSOMES

The following points are made by J.C. Eissenberg and S.C. Elgin (Nature 2005 438:1090):

1) Crack open any cell nucleus and look inside: you will see what look like beads on a string. The beads are nucleosomes, small protein complexes that help to package the DNA (the strings) into the cramped confines of the nucleus. In the past 15 years, nucleosomes have graduated in our understanding from being passive spools for DNA to full partners in the control of genetic information in the cell. Diverse chemical modifications of the histone proteins that form the nucleosome core can alter the expression of the associated genes[1]. These modifications make up what is known as the "histone code", and a major challenge in molecular biology is to decipher how they affect gene expression.

2) Two of the most common modifications are phosphorylation and methylation -- respectively the addition of a phosphate or a methyl group to the amino acids of which the histones are composed. Allis et al[2] previously proposed that reversible phosphorylation of the amino acids serine or threonine in the "tail" regions of histones could antagonize the binding of regulatory proteins to neighboring methylated lysine amino acids, creating a binary control switch. New work[3,4] presents data that strongly support this model and advances our understanding of how histone modification can control chromosome function.

3) Among the many histone modifications that have been described, methylation of lysines and phosphorylation of serines and threonines have attracted much attention. Phosphorylation of the serine at the tenth position in the tails of histone H3 (H3S10) occurs during cell division in eukaryotic (higher) cells. Once the cells have replicated their DNA and begin to prepare for division, nearly all of the histone H3 in the nucleus seems to be phosphorylated at this site. Phosphorylation of H3S10 also occurs at other stages in the cell cycle, but only at discrete chromosomal sites that are associated with gene expression.

4) The methylation of lysines is more complex. Methylation at lysine 9 of histone H3 (H3K9) is found mainly in the heterochromatin -- the dense, mostly inactive regions of the genome. Methylation at lysine 4 of histone H3 (H3K4), by contrast, is associated with active genes. The different outcomes of lysine methylation result from the fact that each modification creates a binding target for a distinct protein. Heterochromatin protein 1 (HP1), which promotes heterochromatin formation (and the consequent gene silencing), recognizes and binds to methylated H3K9 using a region called the chromodomain[5]. CHD1, an enzyme that may destabilize nucleosomes and expose the DNA for gene expression, recognizes and binds to methylated H3K4 through two tandem chromodomains.

5) Fischle et al[3] and Hirota et al[4] examined cells progressing into the metaphase stage of cell division, where the chromosomes become very tightly packed, or "condensed", to facilitate their separation into the future daughter cells. Both groups discovered that these cells accumulated histone H3 that is both methylated at lysine 9 and phosphorylated at the neighboring serine 10. Using antibodies specific for the doubly modified H3, the authors found that this dual modification occurred specifically in heterochromatic regions of chromosomes.

References (abridged):

1. Khorisanizadeh, S. Cell 116, 259-272 (2004)

2. Fischle, W. , Wang, Y. & Allis, C. D. Nature 425, 475-479 (2003)

3. Fischle, W. et al. Nature 438, 1116-1122 (2005)

4. Hirota, T. , Lipp, J. J. , Toh, B. -H. & Peters, J. -M. Nature 438, 1176-1180 (2005)

5. Jacobs, S. A. & Khorisanizadeh, S. Science 295, 2080-2083 (2002)

Nature http://www.nature.com/nature

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GENOME BIOLOGY: STRUCTURE OF DNA IN THE NUCLEOSOME

The following points are made by T.J. Richmond and C.A. Davey (Nature 2003 423:145):

1) DNA in eukaryotic cells is packaged repetitively into nucleosomes by means of extensive association with histone proteins. The hierarchical chromatin structure formed is the genomic substrate relevant to the vital processes of DNA replication, recombination, transcription, repair, and chromosome segregation, and to the pathological progression of cancer and viral disease. Although nucleosomal organization of DNA is essentially ubiquitous throughout genomes and generally repressive to gene expression, it also contributes to gene transcription in a gene-specific manner, suggesting that nucleosome positioning in gene promoter regions is important for genuine gene regulation in vivo. The question therefore arises of how chromatin structure, in which DNA is normally highly compacted, permits site-specific access to regulatory factors and more extensive exposure to the transcription apparatus.

2) The answer is likely to require a knowledge of DNA conformation in the nucleosome core. The core comprises 147 base pairs (bp) of DNA and the histone octamer; compared with the nucleosome, it lacks only 10 90 bp of linker DNA envisaged to be naked or bound to histone H1. The histone-fold domains of the octamer organize the central 129 of 147 bp in 1.59 left-handed superhelical turns with a diameter only fourfold that of the double helix. The relatively straight 9-bp terminal segments contribute little to the curvature of the complete 1.67-turn superhelix. So far, the site-specific regulatory factors that have been discovered bind the linker or terminal regions of the intact nucleosome. The lack of binding to the central region of the superhelix might simply be a consequence of bending the double helix or, additionally, of unusual DNA conformations induced by histone binding. At least one protein, HIV-1 integrase, does prefer DNA bent around the nucleosome in contrast to naked DNA.

3) The initiation of DNA-dependent nuclear processes in the context of chromatin implies that nucleosome position is biased by the DNA sequence to facilitate access by initiation factors. Numerous examples of positioned nucleosomes in gene promoter regions have been described both in vivo and in vitro. Preferential positioning could place factor-binding sequences in nucleosome linker or terminal region DNA. Furthermore, nucleosomes are intrinsically mobile and yield access to their DNA in vitro, allowing even RNA polymerase to transcribe nucleosomal DNA without causing dissociation of the histone octamer. In vivo, energy-dependent chromatin remodeling factors, targeted by gene regulatory proteins and acting directly on the nucleosome core, augment nucleosome mobility. Their mechanism of action most probably derives from the innate ability of nucleosomes to "slide" along DNA without releasing it.

4) In summary: The 1.9-angstrom-resolution crystal structure of the nucleosome core particle containing 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non-histone protein DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fiber formation and chromatin remodeling.

Nature http://www.nature.com/nature

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CELL BIOLOGY: CHROMOSOME CONTACT AND GENE REGULATION

The following points are made by Dimitris Kioussis (Nature 2005 435:579):

1) The cell has evolved many strategies to orchestrate gene activation or repression. New work[1] reveals a novel mechanism of gene regulation, throwing light on how cells organize their genome to respond efficiently to stimuli. The work shows that genes on different chromosomes that are destined to be expressed within a common cell lineage are brought together in the nucleus. Such inter-chromosomal communication has been suspected for some time, but this is the first evidence that it actually takes place.

2) Our understanding of gene regulation has moved from an initial notion of a one-dimensional array of regulatory elements next to each other on the same thread of DNA as the gene to an appreciation that genes are associated with groups of proteins, forming multimolecular complexes that are arranged in structures generically known as chromatin[2]. The subsequent discovery that distant, contiguous sequences can have a profound effect on gene expression introduced a second dimension onto the scene, with "looping" and "scanning" (probably mediated by the attached proteins) invoked to explain these long-range interactions[3].

3) But to explain how genes that are far removed from each other in the genome, and even on different chromosomes, can be coordinated to be expressed together, or to preclude the expression of one another, required a leap into a third dimension. The spatial location of a gene within a cell nucleus can determine whether it is expressed or not: genes residing in areas of chromatin that contain repressive factors (heterochromatin) are silent; conversely, genes in nuclear regions full of activating proteins (euchromatin) are usually switched on[4,5]. How do genes find their appropriate location in the nucleus of a cell, and how are genes that must be expressed herded into active neighborhoods?

4) To address these questions, Spilianakis et al[1] used immune cells called T cells. As they mature, T cells organize themselves into subsets that are assigned specific duties. Thus, T helper (TH) cells produce factors that help other cells of the immune system to function optimally. After antigen stimulation, naive (undecided) TH cells develop into either TH1 cells, which produce one set of effector molecules (for example interferon (IFN)-gamma), or TH2 cells, which produce a different set (for example interleukin (IL)-4 and IL-5). The authors explored the organization of two genomic regions within the TH subsets: the gene encoding IFN-gamma (called Ifng), which is mainly active in TH1 cells, and a multi-gene complex including the genes encoding IL-4 and IL-5 (Il4 and Il5), which is mainly active in TH2 cells.

5) Viewed through two-dimensional analyses, Ifng seems to be regulated by elements found near it on chromosome 10, whereas expression of Il4 and Il5 on chromosome 11 seems to be regulated by a "locus control region" (LCR) on the same chromosome, which directs the entire TH2 gene complex. Spilianakis et al. asked whether these two genetic regions are in close proximity or interact in the nuclei of naive TH, TH1 or TH2 cells. Biochemical and imaging experiments showed that the two regions (on two different chromosomes) are in close proximity in the nucleus of naive TH cells, but after stimulations that induce a TH1 or a TH2 state they seem to move away from each other. The authors' interpretation of this is that in the naive TH cell, the two gene complexes are close together in a region of the nucleus that is poised for gene expression. Upon receiving a specific stimulus, the gene to be activated (for example Ifng after a TH1 stimulus) is allowed to begin expression, whereas its counterpart that is to remain silent (in this case the TH2 genes) is moved, presumably to a more repressed region of the nucleus.

References (abridged):

1. Spilianakis, C. G. , Lalioti, M. D. , Town, T. , Lee, G. R. & Flavell, R. A. Nature 435, 637-645 (2005)

2. Felsenfeld, G. & Groudine, M. Nature 421, 448-453 (2003)

3. De Laat, W. & Grosveld, F. Chromosome Res. 11, 447-459 (2003)

4. Brown, K. E. et al. Cell 3, 207-217 (1999)

5. Kioussis, D. & Festenstein, R. Curr. Opin. Genet. Dev. 7, 614-617 (1997)

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