Fig.5.3 The polytene chromosomes of Drosophila salivary glands. Light microscopy can be used to visualize areas of expansion (puffs, some marked by white bars) and constriction of amplified, polytene chromosomes, associated with active and silent transcription, respectively. DAPI (blue) stains DNA and is brightest when DNA is heterochromatic. Immunofluorescence is used to probe for the presence of a protein complex involved in Drosophila dosage compensation (red). Image kindly provided by Dr. R. Kelley, Baylor College of Medicine, Houston, TX, USA.

The components of chromatin seem fairly simple: two copies each of four proteins, histones H2A, H2B, H3, and H4, 146 base pairs of DNA and a linker histone, HI, if chromatin is more condensed. However, addition of these constituent parts together in a test tube does not lead to self-assembly of the highly organized nucleosome structure. Histones are positively charged due to their enrichment in basic amino acids; DNA is negatively charged. Placing them together in a test tube will cause aggregation and precipitation. An ordered, stepwise assembly process is needed to bring all of the pieces together and form a biologically active, nucleosomal substrate. This challenge must be faced within a cell every time a DNA replication fork passes through chromatin. The component parts must be re-assembled without disruption of overall nuclear structure and with complete integrity and reproducibility to maintain cellular "memory".

Assembly of Chromatin: Chemical Means

The simplest way to avoid nonspecific, histone aggregation and DNA precipitation is to slow down the process of chromatin assembly and give the natural, histone partners, which interact by virtue of their histone fold domains, time to associate with each other: H2A with H2B to form dimers, H3 with H4 to form a tetramer. Two dimers and a tetramer then interact to form an octamer, and DNA wraps around the octamer core approximately 1.8 turns. Slowing the process down can be accomplished in vitro by starting the chromatin assembly process in molar concentration salt solutions with all of the components present, and then diluting the salt over a very long period of time, as much as 48 hours in a cold room. This slow approach toward physiological salt levels gives the histone partners time to associate in a stable, ordered way. This is especially effective with shorter pieces of DNA, approximately 150-200 base pairs in length, to form mononucleosomes, and was employed to assemble the mononucleosomes used in determination of the crystallographic structure (Luger etal., 1997).

Polymers of nucleosomes will form, using the high-salt approach and longer pieces of DNA, but they lack the repetitive spacing of true chromatin. Random clustering of nucleosomes and stretches of bare DNA may result, as there is no specific, DNA binding sequence for nucleosomes. Certain, naturally occurring sequences are considered to have high affinity for nucleosomal assembly, but these enrich 1000-fold or less for a specific nucleosome position. Compared to DNA sequence-specific binding of a transcription regulatory factor, this is a relatively moderate probability and usually nucleosomes assume even these "specific" positions with some random clustering. The enrichment sites, as well as artificial sequences of DNA designed for the specific purpose of higher affinity binding for a nucleosome, are often referred to as nucleosome-spacer sequences (Lowary and Widom, 1998; Thastrom et al., 2004). Plasmids carrying nucleosome-spacer sequences, set approximately 200 base pairs apart, have been developed to use with the simple, high-salt approach to assemble nucleosomes and form repetitive, multi-nucleosome structures. Stretches of chromatin, whether formed in vivo or in vitro, that carry a number of evenly spaced nucleosomes, which form in an ordered manner at specific sites, are referred to as nucleosome arrays.

Well-defined arrays of nucleosomes can be assembled in vitro but these are not truly representative of chromatin found in vivo. The vast majority of chromatin is assembled in a more random, or stochastic, manner to yield a highly repetitive structure but one not generally defined by specific DNA-binding sequences. Specific stretches of chromatin formed by arrays of ordered nucleosomes can occur in vivo, due to some sequence specificity or as a result of DNA binding by transcription factors to set or "phase" nucleosomes at specific positions. Some well-studied examples of positioned nucleosomes, which play an integral role in gene regulation, are the phosphate-responsive Pho5 gene of S. cerevisiae and the glucocorticoid-regulated mouse mammary tumor virus promoter (Archer et al., 1991; Schmid et al., 1992). Positions of nucleosomes within the promoters of these genes are altered in response to environmental factors, which then renders DNA sequences, normally blocked by nucleosomes, accessible to transcription factors and basal transcription machinery. In other cases, positioned nucleosomes enable communication between activating regulatory elements and basal transcription machinery at the core promoter, providing a platform for looping and long-distance interactions in chromatin (Wolffe, 1994).

Assembly of Chromatin: Biological Choices

Within a nucleus there is no option to assemble chromatin in a high-salt solution over a number of hours, nor are protein and enzymatic complexes generally stable under these conditions. Specific proteins, which establish a stepwise order to the process of nucleosome assembly, have evolved to form chromatin in vivo. Histones are found in association with specific chaperone proteins, which escort them to the site of nucleosome assembly and prevent aggregation. For example, histones H2A and H2B are chaperoned by proteins Nap-1/2 and H3/H4 associate with the CAF-1 and ASF-1 proteins. Chaperoned histones are met by protein complexes, which perform the energy-requiring, enzymatic function of nucleosome assembly and spacing. Examples of these assembly factors are ACF1 and its catalytic, ATPase-subunit, Iswilp (discussed below with chromatin remodeling enzymes). As might be expected, these factors come into play especially during DNA replication in vivo, as chromatin assembly occurs efficiently and most frequently in the wake of the processive replication machinery. Energy in the form of ATP is hydrolyzed by specific chromatin remodeling complexes to work with the histones, chaperones and nucleosome assembly factors and form physiological chromatin in vivo (Tyler, 2002).

Biochemical isolation and identification of these factors stemmed from in vitro chromatin assembly using extracts from embryonic sources: Xenopus laevis eggs or oocytes and Drosophila embryos. The building blocks of early development in these, and other, organisms are provided by maternal stores of proteins, membrane vesicles and other components, as supplies for rapid cell division and growth to a point where the developing embryo can rely on its own metabolic processes. These maternal stores can be harvested in the form of cell-free extracts to use in assembly of chromatin in vitro. Any cloned DNA can be added to these extracts and, with the addition of ATP and the right physiological buffers, assembly of the DNA into nucleosomes proceeds over time. Once assembled, the in vitro chromatin must be validated for its physiological relevance by comparison to in vivo chromatin.

The primary point of validation is comparison to in vivo chromatin at the level of first-order structure, meaning the repetitive spacing of nucleosomes along the DNA. The enzyme micrococcal nuclease (MNase) from Staphylococcus aureus digests DNA into nucleotides, in a nearly, but not entirely, sequence-independent way, wherever DNA is not protected by a nucleosome. When limited amounts of MNase are added to chromatin, whether it is assembled in vivo or in vitro, digestion of DNA occurs only within the linker regions lying between adjacent nucleosomes. Complete digestion should yield roughly the mononucleosomal length of DNA, 146 base pairs, plus a few, additional nucleotides due to restricted accessibility of the enzyme. The length of the protected fragment can vary due to the presence of linker histones, such as HI, and sometimes even tightly bound, non-histone proteins. The DNA protected from MNase digestion can be visualized by electrophoretic separation on an agarose gel, which is ethidium-stained or probed as a Southern blot. By titrating the degree of MNase digestion among individual reactions, either by varying enzyme concentration or time of digestion, a "ladder" of DNA, representing protection by mono-, di-, tri-nucleosomes and increasing numbers of nucleosomes, can be seen on these gels with rungs or bands of DNA in increments of 150-160 base pairs (Fig. 5.4). With this standard of effective chromatin assembly, biochemists could then proceed with their attempts to study chromatin regulation in vitro.

How to Activate Chromatin

Regulation of chromatin structure, whether the outcome is repression or activation of gene expression, is due to the combined actions of chromatin remodeling factories, histone modifying enzyme complexes and gene-specific transcription factors, acting in a temporally ordered series of steps. The ground state of any activation process is repression, and this was the first property of chromatin-mediated gene expression that was reconstituted in vitro. Critical DNA elements, including the sites of preinitiation complex assembly and binding sites for activators of transcription, are often occluded or blocked by assembled nucleosomes. Addition of regulatory proteins, such as TBP or specific trans-activators, during nucleosomal assembly allowed "open" or active chromatin formation. This chromatin substrate could be transcribed in a reconstituted transcription system in vitro by addition of basal transcription factors and RNA polymerase II. An "order-of-addition" approach, by which individual general transcription factors, e.g. TFIID, TFIIB, TFIIE, TFIIH, etc, were added before, during or after chromatin assembly, established that TBP or TFIID addition was critical to maintain an open core promoter region and that the general rules of preinitiation complex assembly were followed with chromatin templates as with chromatin-free templates (Rnezetic and Luse, 1986; Workman and Roeder, 1987).

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