Sunday, April 4, 2010

Chromatin Chapter 3: Higher Orders of Chromatin Structure

I don't have an illustration today because (a) I am too lazy to make one, (b) no topic in this post really requires an illustration, and/or (c) I have other things to do.  :)
Higher Orders of Chromatin Structure
The two prior posts concerning nucleosomes and histones compose the first order of chromatin structure, but this packaging alone does not explain how so much DNA is packaged into cells.  Four additional levels have been hypothesized, with varying degrees of evidence for each. 

The second order of chromatin structure is the 30-nm fiber, which consists of six nucleosomes and binds about 1200 bp.  EM images have shown a twisting of the nucleosomes around each other to form a structure that is about 30 nm in diameter when the nucleosomes are in high salt.  There are a few differing models for how the nucleosomes wind around each other.  The first model consists of nucleosomes forming a solenoid, in which they nucleosomes wind around each other in a helix, with six nucleosomes in each turn of the helix.  Histone H1 is also involved in this model, which is the most widely accepted.  The second model is the double-solenoid structure, which consists of two parallel rows of nucleosomes that wind around each other.  Recent evidence via nucleosome arrays have lent more evidence to this model, though this area of research is still active.

The compaction introduced by the 30-nm fiber can result in the condensation of DNA such that it is not available for the proper factors to bind the DNA.  Heterochromatin consists of nucleosomes that condense so highly that the genes contained in the heterochromatin are repressed.  This condensation is is facilitated by heterochromatin protein 1 (HP1), which is a non-histone protein that binds methylated lysine 9 of histone H3 (Me-H3K9).  In contrast, euchromatin consists of DNA that is not repressed and is accessible to factors required for transcription.  This access is facilitated by H2A.Z, which prevents full condensation of the nucleosomes into a 30-nm fiber. 

The third order of chromatin structure is the chromatin loop, which holds fifty 1200-bp fibers, packaging a total of about 60,000 bp of DNA.  These loops are attached the nuclear matrix, at matrix attachment regions (MARs), which promotes supercoiling.  The factors involved in transcription are supercoiled and attached to the nuclear matrix, so those genes in the loops that are transcriptionally active tend to interact with the nuclear matrix as well.  Supercoiling of prokaryotic DNA is performed by DNA gyrases, but eukaryotic DNA gyrases have not been discovered.  Nonetheless, supercoiling does facilitate eukaryotic gene transcription as well.  Further, loops of the chromatin can interact to affect gene regulation.  Studying the interactions of a gene with the nuclear matrix indicates which parts of the DNA are transcriptionally active.  By isolating the nuclear matrix and detecting DNA sequences associated with the nuclear matrix (via probing with radio-labeled 32P dATP), one is able to detect which regions of the DNA are directly associated with the nuclear matrix via MARs.

Initially, it doesn’t make logical sense that active DNA is supercoiled.  However, this is due to the preferential binding of transcription factors to supercoiled DNA.  Additionally, repression factors tend not to bind as well.  The job of removing supercoiling is left to topoisomerases, which cut and reseal the DNA, while unwinding DNA and releasing tension.  Topoisomerase I cuts and re-seals one strand, while topoisomerase II cuts and re-seals two strands.  Additional details about the mechanism of topo II will be discussed in the meiosis posts. 

Real-time fluorescent microscopy has led to a better understanding of the dynamics of chromatin.  While electron microscopy gives a static picture of chromatin, the addition of GFP labels to the chromatin and measuring the movement of chromatin in relation to the nuclear pores (the reference points) has revealed movement of the chromatin that relates to metabolic activity.  During both transcription and chromatin remodeling, the chromatin has been shown to be highly dynamic. 

The fourth order of chromatin structure is called the miniband, which consists of 18 loops, making up a total of one million bp of DNA.  The miniband looks like a helix of loops, with the nuclear matrix inside the helix.  Little else is known about it, other than it is highly condensed.

Finally, the fifth order of chromatin structure is the chromosome, which we know consists of roughly 75 million bp of DNA, depending on which chromosome you consider.  The structure of the chromosome is interesting in its own right, and there are a number of features to be discussed.  The telomeres of the chromosomes consist of tandem repeats of the sequence TTAGGG, up to 15 kbp.  The telomeres cap the ends of the chromosomes and indicate the replicative capacity of the chromosome.  The telomere hypothesis posits that cells become senescent at a threshold telomere length, meaning that cells have a finite number of divisions.  Telomeres shorten with each chromosome replication (and cell division) due to the inability of the cell to replicate the linear ends of the chromosomes.  Telomeres in sex cells are long because they replicate frequently, while somatic cells that do not actively replicate have short telomeres.  Therefore, the telomere length and its regulation make up the major aging mechanism in the cell.  Those cells that will divide many, many times express the protein telomerase, which is involved in lengthening the telomeres at the ends of the chromosomes by providing an RNA template and polymerase function. 

One can stain different regions of chromosomes to obtain an idea of how actively transcribed it is.  Giemsa staining and visualization by light microscopy is the most frequently used method.  The chromosomes are trypsin digested and stained with Giemsa.  G-light areas, which are areas that the Giemsa does not stain well are considered unfolded and relaxed.  The genes in these G-light bands are susceptible to radiation and are often oncogenes.  Additionally, these genes are usually constitutively active (housekeeping genes).  In contrast, G-dark bands are not sensitive to trypsin digestion and appear dark when stained with Giemsa.  The genes in these bands are considered tissue-specific and are replicated later during S-phase.  Finally, C-bands (for constitutive heterochromatin) remain condensed and are also dark when stained with Giemsa.  The areas of the chromosome found in C-bands replicate late in S-phase and may consist of telomeres, the centromere, and satellite DNA.  

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