Chromatin Structure and Superstructure

Chromatin Structure and Superstructure

            Essentally all the DNA in eukaryotic nucleus is complexed with histone in a basic 10 nm diameter chromatin fibre, which may be fruther folded into a 25 – 30 nm diameter fibre. The basic fibre (or nucleous filament) is a linear array of connected nucleosomes which about each other. Each nucleosome contains about 200 base pair (bp) of DNA (166-241 bp depending upon source) assosaited with an octameric protein core comprising pairs of each other of four types of histone-the lysine-rich histone H2A and H2B, and the arginine-rich histone H3 and H4 which have been particularly well conserved during evolution. One molecule of the fifth histone, H1, is associated with the outside of each nucleosome, binding partly to linker DNA between nucleosome cores.

            There have been several recent reviews of the nucleosome ⁵³ and of chromatin more generally^41,77, so no attempt is made here to be comprehensive. Rather some aspects of chromatin structure that are currently of particular in interest  will be discussed. These include the detailed structure that are currently  of particular interest will be discused. These include the detailed structure of the nucleosome and its placement has any functional significance over and above its structural significance as a parking unit. Specifically, is there ‘phasing’ of nucleosome and DNA sequences? The nature of the next level of folding of the nucleosome filament will be discussed; the stability of such higher order structure could in principle determine whethera particular region of chromatin is rendered accessible for transcription, or masked and inaccessible. Finally our current view of the structure of transcriptionally active chromatin chromatin, recentially reviewed elsewhere^41,76,124, will be summarized.

 

 Figure 1. Structure Chromatin and Activity

 

Structure of nucleosome                   

An outline

            The nucleosome filament shows periodic diffrential susceptibility, to several endonuclease e.g. endogenous nuclease, micrococcal nuclease, DNase II. Evidently as the DNA leaves the surface of one histone octamer and passes to the next it becomes more sensitive to these enzyme than the DNA protected by hitone in the core, and nucleosomes or oligonucleosome are released as a result of double strand cuts in these regions of ‘linker DNA’. Mononucleosome initially contain a full repeat length of DNA (e.g 200 bp in rat liver). Continued digestion of these particles by micrococcal nuclease, acting as an exonuclease , reveals barriers to fruther digestion, (presumably) imposed by histone-DNA interactions. The first such impediment arieses when the DNA is trimmed from its full repeat length (e.g 200 bp) to 166 bp, which occurs without any change in histone composition of the particles. A more substanstial barrier aries when the DNA is trimmed to 146 bp (originally asigned as 140 bp), giving a particle depleted of H1, which is a metastable intermediate in digestion and protected from fruther attack by the octameric histone core. This paticles isolated at the two stages of digestion have been termed chromatosome (166 bp) and nucleosome core particles (146 bp). The digestion of the nucleosome by micrococcal nuclease can therefore be summarized as follows :

Nucleosome                            Chromatosome                        Nucleosome core Particles

     200 bp⁺                                    166 bp                                          146 bp

         +                                                 +                                              +

    octamer                                      octamer                                    octamer

         +                                                 +                                              +

        H1                                               H1

            All three particles sediment about 11-12S. Analysis of their DNA sizen reveals that the broad band observed for intact nucleosome (width ~60 bp) is sharpened during digestion so that the nucleosome core particles DNA content is much less heterogenous (146±6 bp). This relative homogeneity in DNA content is an important factor in archieving the crystallization of core particles (see below). An external location of the DNA in the nucleosome is indicated both by the accestability of DNA along its whole length to DNase I⁸³, and by nueron scattering studies^37,86. The nicking by DNase I at intervals of about 10 nucleotides along each strand is taken to reflect the periodicity of B-from DNA wound around a histone core (see also next section).

 

 Figure 2. Histone H1

 

            x-ray diffraction by single crystals, combined with electron microscopy of crystal, has shown the 146 bp core particles to be a slightly wedge-sharped disc, 11nm in diameter and 5.5 nm high, containing about 1.75 turnx of DNA with about 80 bp per tun²⁹. The simplest assumption, in the absence of evidence to the contrary, is that DNA duplex is smoothy bent around the histone core in a regular superhelix which is left-handed^32,71. The presence of a dyad axis in the nucleosome core particle has been demonstrated uniquevocally by higher resolution X-Ray diffraction study²⁸, and by neutron diffractiom by single crystals¹¹.

            Extension of the 1,75 turns if DNA in the 146 bp nucleosome core particle by 10 bp each andwould give a particle containing two complete turns of DNA. The corresponds to the 166 bp chromatosome are trimmed to core particles suggests that H1 binds to the 10 bp extensions at the ends of the core particle. The presence of H1 gives the nucleosome filament a zigzag apperance in electron micrographs, in contrast with a beeds-on-a-string apperance when H1 is absent, and prevents the unfolding of nucleosomes at very low ionic strength (˂0.2 mmol/ℓ), at least on microscope grid¹¹². The conculsion drawn from all these observation is that H1 seals two complete turns of DNA around the nucleosome and fixes and the entry and exit points close together.

            H1 has three distinct regions of amino acid sequence¹²⁰  which appear to correspond to three ‘domains’ of structure, namely a central globular region of about 80 residues, which is relativity conserved in amino acid sequence from species to species, flanked by two very basic regions which appear to be flexible and not tightly folded, at least in solution³⁶. The globular portion, isolated after tryptic removal of the flanking regions, seems to be sufficient to restore to H1-depleted chromatin a pause in the digestion at 166 bp³, suggesting that this domain of H1 binds to the two terminal 10 bp regions in the chromatosome, closing two superhelical turns of DNA. For condenstation of chromatin the basic flanking regions are required, and these probably interact with the linker DNA, in a manner as yet unclear.

            Both the nucleosome core particle and the core histone octamer prossess a dyad axis symmetry, raising the possibility that there may two binding sites for H1. Although the number H1 molecules per nucleosome has been in some dispute, recent measurements have shown that nuclei from several sources contain only sufficent H1 for one H1 per nucleosome on average.

 

The Linking Number Paradox

            The paradox is that although X-ray analysis (see above) shows that threre are two superhelical turns of DNA around the nucleosome, the change in linking number of closed circular DNA extraced from SV40 minichromosomes is only -1.25 per nucleosome. The change in linking number (where the linking number is essentially the number of times one strand of DNA duplex crosess another)  aries from some combination of change in the twist and writhe of the DNA. The number of physical superhelical turns of DNA around the nucleosome (essentially, the writhe) will therefore equal of change in linking number only if the helical periodicity of the DNA (i.e the twist) is the same for DNA free in solution and DNA associated with histone in the nucleosome. It was pointed you out²⁹ that the paradox might be resolved if the helical periodicity was reduced from 10.5 bp per turn in solution to 10.0 bp on the nucleosome, the letter being the value suggested by the DNase I digestion pattern (see above), other proposals have also made for resolution paradox^106,131.

            DNA in solution has subsequently been found, by two independent methods of measurement^{94, 122}, to have an average helical periodicity of about 10,6 bp per turn. It would therefore seem that the linking number paradox disappears. However redetermination of the distance between DNase I cutting sites in chromatin gave a value of 10.4 bp on average.  Thus the peridiocity of cutting by the DNase I on the nucleosome essentially the same as the helical peridiocity of DNA in solution, and the linking number paradox remains. However, it has been argued^{48, 91} that the nuclease cutting peridiovity of 10.4 bpon the nucleosome is compatible with a helical repeat of 10.0 bp since acces of the relatively large enzyme (DNase I) to the DNA duplex will be restricted by the other superhelical turn of DNA, so that the enzyme does not cut that the outherwise maximally exposed phosphodiester bonds. This explanation would remove the linking number paradox, since the helical peridiocity of the DNA (the twist) changes, on binding to histone octamer, from 10.6 bp to 10.0 bp per turn. The X-ray diffraction patterns from nucleosome core particle crystals do indeed show strong intensities at 0.34 and 1.2 nm characteristic of B-from DNA with 10.0 bp per turn.

 

Eukaryotic Gen

EUKARIYOTIC GEN

Introduction

            Since 1953, when Crick, Watson and Wilkins provided insight into the double helical structure DNA, the steady snowfall of new knowledge about genes and gene structure has turned into a blizzard, a blizzard that all involved in genetics most endeavour to live with and work in. This book sets out to make life amidstthe rapid accumulation of new knowledge easier to mark a road through the snow. To do so is ambitious and beyond the skills of single individual. What we have therefore soughtto do has been to compile a book, with the chapters written by workers who can talk authoritatively in their field, that providing up to date information in a formal which reflects the comparative importance of different levels of information in terms of gene suquences, chromatin and chromosomes.

 

Figure 1. Picture Watson and Crick Explain DNA Model

            How can the present blizzard of the new information be accounted for? Essentially it result from the effeciency but comparative simplicity of the genetics code, consisting as it does of triplet codons of only four different bases and coding for little more than twenty different bits of translated informations. This coding simplicit, together with the coplementary nature of the squence on the two sister strands of the DNA helix, implies that if ways could be found to locate gene sequence with chromatin and determine base sequence within be isolated gene, then it would be possible to begin a study of genetics from precisely the opposite and to that which had occupied man’s attentions over the centuries. Instead of studying phenotype, familiar inhertance or at best, the expression of genes in their constituent proteins it has rather suddenly become possible to read out the genetic code itself and attempt to unscramble what is significant in the message so revealed. While it must be acknowledged that interpreting the facts about inhertance from knowledge of DNA squence is not by any means straightforward, and that many aspects of genetics will only be effectively understood by knowing that whole story from gene squence to phenotyphic character, yet it cannot be denied that the new approach has introduced a major revolution in the study of genetics and that our understanding of the processes of inhertance has made a considerable advance.

New technology has provided new insight

            It is wrothwhile to consider what the new techniques have been which have proved so valuable in genetics. Some of the technology is discussed in greater detail in the introduction to individual sections of this book, and to a lasser extent in some of the chapters themselves. But here it apropriate to consider the range of tchniques which have found application at many different levels in the genetics framework.

1.    At the level of the chromosome

Although the chromosomal location  of genes had been assumed since the turn of the century and known with certainty for a few decades, chromosomes have proved difficult to study. In most cells they were only visible during mitosis and many looked superficially similiar. Even the chromosomal location of large blocks of genes could not be readly ascertained as is revelead by the early ambiguity about the chromosomes involved in human mongolism. The great step forward came with chromosome banding. Following on from the application of fluorescence technique to the staining of chromosomes, it became apparent that particular cytochemical methods involving strong alkali or formamide would also specifically stain the centromeric chromatin. As such C banding became commonplaces; it also became clear that somewhat smiliar techniques, when followed by Giemsa staining, would reveal a banded pattern over the entire chromosome, called the G band pattern. What was particularly useful was that different chromosomes displayed different G band patterns , so permiting for the first time uniqeuivocal identification of particular chromosomes.

 

Figure 2. Chromosome 

Ascribing a gene to a particular chromosme or even a unique locus on a chromosomes remains difficult. Such assigment of genes has traditionally depend on linkage studies and measuremeant of cross-over frequencies where possible, but there are now staining techniques which in at least some circumstances, can reveal the location of spesific genes e.g the use of silver stain to localize active ribosomal RNA sequences. Genes which are repetitious and occur in large blocks such as the transfer RNA genes can be localized by autoradiography following in situ hybridization with radioactive cDNA. Some works has been carried out the location of unique structural gene sequences by autoradiography but the weak signal obtained renders the interpretation of such experiments with complementray cDNA extremely difficult. A quite different technology which can permit unequivocal assignment of a particular structural gene to an individual chromosome is that of cell fusion and the formation of bispesific hybrid cell. In particular by the exploitation of Sendai-virus medicated cell fusion between mouse and human cells, followed by the culture of the hybrids, often with of gradual loss of most of human chromosome set, cell lines have been obtained which the contain an’all-mouse’genome with the exception of a single human chromosome. Banding of the chromosome reveals the identity of the single human chromosome while a prolonged assay for spesific protein expression, say the timidyne kinase gene, permits unequivocal assignment of a particular structure gene to an individual chrosome.

 

Figure 3. DNA in Chromosome

            Useful as these advances in whole chromosome technology have been, they are certainly less startling than the developments in the fields of chromatinand actual gene sequence, and these latter areas are chiefly what this volume seeks to consider.

2.    At the level chromatin

Early theoretical attemps to understand the physical relationship betwwn histone and DNA formed an inner core and that the histone molecules, applied to the surface of the double helix could be regarded as the outher sheath. The true structure of chromatin was initially indicated by the simple but crucial experiments of Hewish and Burgoyne. These Australian workers digested rat liver chromatin with an endogeneous calcium active nuclease enzyme and produced the now familiar 200 base pair repeat ladder on electrophoresis of the DNA fragments from the digest. This result clearly implied that the DNA was in some way differentially protected from digestion, execpt for certain exposed sites located some 200 base pair apart. When this observation was put alongside the results of chromatin fine structure analysis in the electron miscroscope, the nation of histone bead as repeating structure in chromatin soon emerged. Nuclear magnetic resonance (n.m.r) studies soon esrabilished that, contrary to the earliest assumption about histone/DNA conformation, the DNA was wound on the outside of histone octamer. So was conceived what is now accepted as an accurate pictures of the subunit of most chromatin, the nucleosome. Some of the earlier chapters in this book provide an up to date view of nucleosome and how its structure is implicated with such other aspects of genetics as gene regulation and the arragement of chromatin chromosomes.

Its now clear the nucleosome are the invariable chromatin subunit in all transcriptionally inactive chromatin (expectsperm) and probably in most active chromatin as well. Arragement of the nucleosome in the higher oreder structure of chromatin is less clear but already exploitation of techniques such as nuclear magnetic resonance have provided persuasive evidence that nucleosome are arranged as helical solenoids, phraps each solenoid representing a loop or domain of DNA attached to backbone or scaffold in the intact chromosome. 

 

Figue 4. DNA in Chromatin

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