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Chromatin, Chromosomes, and Genome Integrity
We have a strong community of labs in the Department of Biology that focus on various aspects of chromosome biology, including gene regulation, DNA replication, chromosome segregation, chromosome structure, the DNA damage response, and the maintenance of genome integrity. Our labs use a variety of model systems to study chromosome biology including budding yeast, plants, worms, fruit flies, mice, and human cells. Our major interests include how chromosomes are duplicated in S phase and segregated in M phase, how DNA breaks promote crossovers in meiosis, how DNA damage is repaired, the role of mutations in evolution, and how epigenetic modifications of chromatin regulate the cell cycle, gene silencing, and cancer.
There is significant interaction and exchange of ideas among the labs studying chromosome biology. We also have excellent core facilities to support our research, including state-of-the-art facilities for microscopy, genomics, Drosophila genetics, and biochemistry. The collaborative community and wonderful facilities make IU Biology a great place to study chromatin, chromosomes, and genome integrity.
What is chromatin?
Chromatin is the genetic material found in a eukaryotic cell during the interface. That is, before cell division. It is formed by bilaterian DNA, associated with basic proteins rich in amino acids such as arginine and lysine. Two types of chromatin can be distinguished during the interface:
-The euchromatin: where is little condensed chromatin.
-The heterochromatin: where chromatin so condensed and stained by 90%. In it, the DNA is not transcribed and remains inactive.
1.5: Chromosomes and chromatin - Biology
Packed inside the nucleus of every human cell is nearly 6 feet of DNA, which is subdivided into 46 individual molecules, one for each chromosome and each about 1.5 inches long. Collecting all this material into a microscopic cell nucleus is an extraordinary feat of packaging. For DNA to function when necessary, it can't be haphazardly crammed into the nucleus or simply wound up like a ball of string. Consequently, during interphase, DNA is combined with proteins and organized into a precise, compact structure, a dense string-like fiber called chromatin, which condenses even further into chromosomes during cell division.
Each DNA strand wraps around groups of small protein molecules called histones , forming a series of bead-like structures, called nucleosomes , connected by the DNA strand (as illustrated in Figure 1). Under the microscope, uncondensed chromatin has a "beads on a string" appearance. The string of nucleosomes, already compacted by a factor of six, is then coiled into an even denser structure known as a solenoid that compacts the DNA by a factor of 40. The solenoid structure then coils to form a hollow tube. This complex compression and structuring of DNA serves several functions. The overall negative charge of the DNA is neutralized by the positive charge of the histone molecules, the DNA takes up much less space, and inactive DNA can be folded into inaccessible locations until it is needed.
There are two basic types of chromatin. Euchromatin is the genetically active type of chromatin involved in transcribing RNA to produce proteins used in cell function and growth. The predominant type of chromatin found in cells during interphase, euchromatin is more diffuse than the other kind of chromatin, which is termed heterochromatin . The additional compression of heterochromatin is thought to involve various proteins in addition to the histones, and the DNA it contains is thought to be genetically inactive. Heterochromatin tends to be most concentrated along chromosomes at certain regions of the structures, such as the centromeres and telomeres. Genes typically located in euchromatin can be experimentally silenced (not expressed) by relocating them to a heterochromatin position.
Throughout the life of a cell, chromatin fibers take on different forms inside the nucleus. During interphase, when the cell is carrying out its normal functions, the chromatin is dispersed throughout the nucleus in what appears to be a tangle of fibers. This exposes the euchromatin and makes it available for the transcription process. When the cell enters metaphase and prepares to divide, the chromatin changes dramatically. First, all the chromatin strands make copies of themselves through the process of DNA replication. Then they are compressed to an even greater degree as they undergo a 10,000-fold compaction into specialized structures for reproduction, the chromosomes (see Figure 2). As the cell divides to become two cells, the chromosomes separate, giving each cell a complete copy of the genetic information contained in the chromatin.
The number of chromosomes within the nuclei of an organism's cells is a species-specific trait. Human diploid cells (those that are not gametes) characteristically exhibit 46 chromosomes, but this number can be as low as 2, as is the case for some ants and roundworms, or more than a thousand, as exemplified by the Indian fern ( Ophioglossum reticulatum ), which has 1,260 chromosomes. Accordingly, the number of chromosomes a species has does not correlate to the complexity of the organism.
Chromatin and Chromosome Biology
The genome is the blueprint of life. It is a set of DNA molecules that contain all of the instructions for an organism&rsquos development and the ability to respond to the environment. Every cell contains the same blueprint but depending on how these instructions are executed will lead to a variety of cell types (such as blood cells, neurons, and muscle cells) and ultimately a complex organism. This is a tremendous amount of information and in fact if each molecule of DNA was aligned end to end it would span 2 meters (
6.5 feet). Yet amazingly, these molecules all fit into a cell nucleus that is only 0.000006 meters in diameter.
The packaging of the genome into the cell nucleus is accomplished through chromatin. Chromatin is the complex of genomic DNA with proteins called histones, where each histone-bound DNA molecule is referred to as a chromosome. However, chromatin not only compacts the genome into the nucleus, but is also the mechanism controlling how the genome is read out from cell to cell. Thus, chromatin is often referred to as the epigenome (&ldquoover&rdquo the genome). Chromatin is incredibly dynamic, reorganizing during development to establish cell-type, as well as in response to an array of environmental stimuli. Importantly, dysregulation of chromatin underlies a number of diseases including developmental disorders, cancer, heart disease, neurological disorders, and more.
Research on chromatin and chromosome biology is aimed at understanding exactly how the genome is packaged into chromatin and the myriad of ways in which it is dynamically regulated. Our team of scientists employs a range of experimental and computational approaches to study this. We are investigating exactly how the histone proteins are organized from cell to cell, how chromatin is regulated by the environment, how genomic information is read from chromatin by the molecular machinery, and how chromatin dysregulation leads to disease.
The genomics revolution
The third theme underlying many talks was genomics. Although the Saccharomyces cerevisiae genome sequence was completed several years ago, the majority of talks addressing chromosome function still relied on classical yeast biology. Having the genome sequence has not altered the major task of identifying and characterizing mutants. Instead, like PCR, genomics gives us new tools that enormously speed up the process of data collection it complements, rather than replaces, more classical biological approaches. Mike Snyder (Yale University, USA) described several approaches that are producing valuable data for the community (Yale Genome Analysis Center [http://ygac.med.yale.edu/]), including the effort to transposon-tag each budding yeast gene with a hemagglutinin (HA) tag to allow analysis of subcellular localization of 1,200 nuclear proteins studied, 600 were found to bind chromosomes. Another project is based on the 'ChIP chip' method: chromatin immunoprecipitation against chip arrays. Transcription factors can be HA tagged, immunoprecipitated while bound to chromatin, and the bound sequence can be hybridized to a microarray of intergenic regions, to identify putative target genes. In an analysis of binding by the Swi4p transcription factor, some binding regions were identified that lacked the canonical Swi4p binding sites. Over 160 binding sites in intergenic regions were identified 40% of the sites neighbored open reading frames (ORFs) that exhibited G1/S periodicity, and over half of these ORFs were of unknown function.
Snyder also described analysis of 106 of the 122 protein kinases encoded by the budding yeast genome. Microarray wells are coated with a potential substrate and a purified kinase is added to each well for a kinase assay, allowing a fingerprint of substrate specificity. The protein kinases were assayed against various substrates, such as casein, histone H1, and poly(TyrGln). Most kinases appeared to be promiscuous, with only half specific for one or two substrates. These approaches demonstrate the utility of having the genome sequence, but the interpretation of the data obtained requires a return to the organism to determine their relevance using classical means, including analysis of protein or substrate interactions and mutant analyses.
Tim Hughes (Rosetta Inpharmatics, Kirkland, USA) described progress on the use of expression profiles as a method to probe genes of unknown function. In S. cerevisiae, roughly 1,800 of the 6,000 genes remain uncharacterized. Rosetta Inpharmatics suggests that the whole genome expression profile is distinct when a given gene or pathway is disrupted, so particular expression profiles can become diagnostic of particular pathways. By cluster analysis of expression profiles, they can therefore classify novel gene functions as related to known pathways. An unexpected finding was that approximately 8% of deletion mutants exhibited some level of aneuploidy. For example, the deletion strain that has lost the ribonucleotide reductase subunit Rnr1p can apparently survive by maintaining an extra copy of chromosome IX, which harbors RNR3 (a known dosage suppressor of rnr1). M.K. Raghuraman (University of Washington, Seattle, USA) finished this chip-related discussion by describing genome-wide analysis of DNA replication origins in yeast. Defining budding yeast replication origins by combining classical density gradient analysis of replicated DNA and microarray analysis, Raghuraman and colleagues reported the identification of 376 replication origins in the yeast genome and characterized their replication timing. Peter Sorger (Massachusetts Institute of Technology, Cambridge, USA) described an 'image informatics' approach that relies upon a large database of biological images, in this case three-dimensional images of live budding yeast cells carrying centromeres tagged with green fluorescent protein (GFP), and uses computational analysis to identify factors affecting kinetochore function. With this technique, Sorger demonstrated that sister chromatids exhibit transient separation during prometaphase in the absence of cohesin proteolysis, and suggested that the yeast kinetochore acted as a tensiometer, detecting the tension between the microtubules and the chromosomes.
All living organisms are made of cells. Some organisms are single-celled and spend their life made of just one cell and some organisms, like humans, are multi-cellular (have many cells). Here are some basic points that you always have to remember about cells:
- cells are the structural and functional units of all living things
- all cells come from pre-existing cells
- cells contain hereditary information (which is passed from "parent" cells to "daughter" cells during cell division).
Cells are often called "the building blocks of life". Not only are all living things made of cells (the 'building block' part), they also are living functional units themselves. This is because cells do the following:
- obtain nutrients and other molecules through the cellular membrane (or cell wall) and convert that to biologically useful energy and useful molecules for building new cells (such as proteins and nucleic acids)
- make more cells (reproduction)
Types of cells
There are two types of cells: prokaryotic and eukaryotic. Prokaryotes include bacteria and blue-green algae. These are single-celled organisms in which the genetic material is not distinctly separated from the other components of the cell. Eukaryotes include everyone else. These can be single-celled, but are more often multi-celled organisms in which the genetic material is separated from all of the other cellular components (by something called the nucleus). Because our focus is on humans, the rest of our discussion will center on eukaryotes and eukaryotic cells.
Figure (PageIndex<1>) - A typical animal cell and its parts
Each animal cell consists of a selectively permeable membrane (plants have cell walls) which contains the cytoplasm.
Organelles are like "little organs" within the cytoplasm, each performing a different vital cellular function. These are some of the more important (to us) organelles.
- Mitochondria: These are the "powerhouses" of the cell, responsible for production of the energy-rich molecule, ATP (adenine triphosphate), which powers the activities of the cell. Each cell has hundreds to thousands of mitochondria. Mitochondria have their own DNA, known as mitochondrial DNA (mtDNA), which is different from nuclear DNA (see below). Mitochondrial DNA has become an important tool in evolutionary research.
- Endoplasmic reticulum: This is a transport network for molecules that have specific destinations or require certain modifications. It comes in two types: smooth and rough. Rough endoplasmic reticulum has ribosomes (see below) on the surface.
- Golgi apparatus: Processes and packages large molecules such as proteins and lipids (fats) that are produced by the cell.
- Lysosomes: Breaks down non-usable organelles, food, viruses, and bacteria using enzymes.
- Ribosomes: Protein synthesis
- Nucleus: This is where you'll find the hereditary material, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
In this interactive exercise, you can identify the parts of a typical animal cell and review the functions of the various organelles.
DNA (deoxyribonucleic acid) is the genetic blueprint of the cell. The structure of DNA is what's known as a "double helix", which looks much like a twisted ladder. The basic unit of DNA is a molecule called a nucleotide. Nucleotides are made of a sugar (deoxyribonucleic acid), a phosphate group, and a nitrogenous base. There are 4 different types of nucleotides, depending on what type of nitrogenous base they have.
Figure (PageIndex<2>) - DNA structure
These are separated into 2 groups:
On the DNA "rungs", the bases must go in pairs: adenine (A) always bonds with thymine (T) and cytosine (C) with guanine (G). So, for base-pairs remember it's always A+T and C+G! (That rule is sometimes called "The base-pair rule".) If you want to explore further, check out what the Human Genome Project has learned about the human DNA sequence
RNA (ribonucleic acid) has a very similar structure to DNA, except that it's single-stranded, has a different sugar (ribonucleic acid, rather than deoxyribonucleic acid), and does not contain thymine (T). Instead, it contains uracil (U), which like thymine, bonds with adenine (A). So, in this case, the base-pair rule is A+U and C+G.
For our purposes, we will concern ourselves with two of DNA's functions: replication and protein synthesis.
In this case, the goal is to replicate a DNA double helix to produce more DNA. DNA replication occurs in the nucleus of the cell.
The basic steps of DNA replication are:
1) The DNA is "unzipped" into two single strands.
2) Each unzipped strand acts as a template for reproduction of the complimentary strand.
3) The product is two copies of the original DNA, each containing one strand from the original DNA and one strand that has been added on.
Here's a slightly more thorough illustration of the process: DNA replication - simple.
And for those who are really curious about the details of the process: DNA replication - complex.
And for those who are looking for a little fun with their DNA replication: DNA - the double helix game
Important note: The important thing to remember about DNA replication is that when the complimentary strand is created, sometimes accidents or mistakes happen. For example, a G gets matched up with a T instead of the proper A. This is where some of the variation in hereditary information comes from.
Figure (PageIndex<3>) - Protein synthesis
Proteins are the most common large molecules in cells. Bones, muscles, and red blood cells (among lots of other body parts) are made mostly of proteins. Therefore, the production of proteins is obviously a very important process in the body.
Proteins are made of smaller units called amino acids. There are 20 different amino acids, 9 of which are "essential amino acids", which means that they must be consumed through the diet, rather than being synthesized by the body. The sequence of amino acids in a protein determines its structure and function.
The base pair sequence of the DNA molecule is known as the genetic code. The genetic code consists of 3-base sequences called codons.(Remember our friends A, T, G, and C -- the nitrogenous bases? That's what we're talking about here.) Each codon either codes for an amino acid, or signals that the protein chain is starting (an initiation codon) or stopping (a termination codon). The table on the right shows which codons code for which amino acids.
Each DNA molecule contains the information to make up many different proteins. A portion of a DNA molecule responsible for making up a single protein (or sometimes just part of a protein, called a polypeptide) is a gene. Therefore, each DNA molecule consists of many genes, that code for many proteins.
Protein synthesis has two basic steps: (1) transcription and (2) translation.
1) The gene (DNA) is copied onto RNA. The RNA copy of the gene is called the messenger RNA (or mRNA).
2) The mRNA leaves the nucleus and goes to the rough endoplasmic reticulum. (Remember this organelle from the cell structure section above?)
1) The mRNA goes into the ribosomes, where tRNA (translation RNA) reads the mRNA.
2) As the tRNA reads the mRNA, it attaches complementary amino acids to the newly synthesized amino acid chain (AKA the growing protein chain).
Important note: The important thing to remember about protein synthesis is that when a protein is synthesized, sometimes accidents or mistakes happen. This is where some of the variation in protein synthesis comes from. Additionally, remember the possible mistakes in DNA replication? If the original DNA strand is changed so that a gene is altered, that can affect protein synthesis as well.
This video has fantastic digital animations illustrating DNA replication and protein synthesis.
Chromatin and chromosomes
Recent advances in our understanding of chromosome behavior have been made possible by the development of cutting-edge cytological and molecular techniques. Presentations in this Minisymposium applied these techniques to a diverse set of topics.
Sarah Elgin (Washington University) described her lab’s studies on the 1360 transposable element in Drosophila melanogaster. Previously, they showed that transgenes possessing this element promoted heterochromatin formation when inserted at ectopic sites in the genome. Now, using site-specific integration technology, they performed structure/function analyses and mapped the region responsible for silencing to the transposon’s transcription start sites. She also discussed her continued studies on the connection between heterochromatin proteins and RNA interference machinery. Knockdown of Piwi, an Argonaute/Piwi family member that binds Piwi-interacting RNAs and Heterochromatin Protein 1a (HP1a), in the female germline caused loss of HP1a and H3K9me2/3. This resulted in increased expression of the telomeric element HeT-A, demonstrating a role for Piwi in gene silencing in the female germline.
Yasushi Hiraoka (Osaka University) identified the Schizosaccharomyces pombe Sme2 locus as a chromosome-pairing site during meiosis. A genomic fragment of Sme2 containing the TATA box and transcription start site is both necessary for pairing at the endogenous location and sufficient to induce pairing at ectopic genomic sites. Sme2 encodes a noncoding RNA that remains associated with the locus, possibly serving as a scaffold for proteins involved in pairing.
Kristine Willis (Georgetown University) spoke about the relationship between gene activation and positioning within the Saccharomyces cerevisiae nucleus. By tracking both the position and the protein product of an inducible reporter gene, she finds that the reporter gene requires movement to the nuclear periphery before activation. In a subset of the cells, the activated gene remains at the periphery, while in other cells the gene moves to the interior and remains active. Interestingly, mutations in chromatin-remodeling factors caused defects in retention at the periphery, suggesting a requirement for a remodeled chromatin state at the periphery.
Using S. cerevisiae as a model to study chromosome segregation, Min-Hao Kuo (Michigan State University) discovered that histone H3 monitors mitotic tension between sister chromatids at pericentromeres. Mutation of several amino acids within the H3 “tension-sensing motif” caused the loss of Shugoshin (Sgo1) from pericentromeres and chromosome segregation defects. GCN5, a histone acetyltransferase, suppressed the Sgo1 recruitment and segregation defects. A model was proposed whereby GCN5 acts as a negative regulator of Sgo1 spreading, limiting localization to the pericentric region.
Carl Schildkraut (Albert Einstein College of Medicine) used single-molecule analysis of replicated DNA to examine replication forks and origins. Studies of replication forks stalled by hydroxyurea revealed that phosphorylation of replication protein A reduced single-stranded DNA and stimulated DNA synthesis to maintain replication fork integrity. When applied to studies of mouse telomeres, this technique revealed that chromosome ends replicate bidirectionally from sites surrounding and within telomeric repeats.
Deborah Lannigan (University of Virginia) identified a function for the extracellular signal–regulated kinase 8 (ERK8) in DNA replication. ERK8 shares sequence identity with mitogen-stimulated kinases and was hypothesized to have self-phosphorylating activity, yet its role in cell biology was unknown. Knockdown of ERK8 reduced levels of proliferating cell nuclear antigen (PCNA), resulting in increased DNA breaks. Adding back excess PCNA rescued this defect. Substitutions within the phosphatidylinositol phosphate (PIP) domain of ERK8 recapitulated the ERK8 knockdown phenotype, suggesting this domain as a site of interaction. Knockdown of HDM2, an E3 ligase with a PIP domain, increased the levels of PCNA and rescued ERK8 knockdown defects, suggesting that recruitment of ERK8 to chromatin protects PCNA at the replication fork.
Collectively, these presentations introduced American Society for Cell Biology Annual Meeting attendees to the latest technologies used to reveal fundamental principles that govern chromosome biology.