Friday, April 23, 2010

More about the Nucleus: Matrix, Envelope, Pores, Lamins

I needed that little break.  Exams have calmed temporarily and I have begun studying in earnest yet again.  Now I plan to take a slightly different approach with these updates by moving through the material chronologically, as it was taught.  Maybe using this method, I'll be able to refer back  and interlink posts more efficiently.
The Nuclear Matrix
As mentioned, DNA wrapped in nucleosomes loops and attaches to the nuclear matrix, but what exactly is the nuclear matrix?  Technically, the nuclear matrix consists of what is left after the DNA, lipid, and protein content of the nucleus has been cleared.  It consists of the nucleolus, the nuclear pore complex and lamina, and the internal nuclear matrix.  Many have argued that the nuclear matrix is simply an artifact of the extraction process.

The nucleolus in the nuclear matrix consists of ten chromosomes that converge to form a small compartment.  These ten chromosomes all contain genes for rRNA, and the nucleolus is where the rRNA is synthesized.  The ribosomal rRNA is synthesized by RNA pol I and III, and after synthesis, proteins are added to the rRNA while it is still in the nucleus.  Various subcompartments of the nucleolus have also been identified: the fibrillar center consists of the nucleolar organizer and the rDNA genes; the dense fibrillar (pars fibrosa) consists of the sites of transcription; and the granular (pars granulose) makes up the ribosome subunits. 

The Nuclear Envelope and Pore Complex Lamina
The nuclear envelope consists of a double membrane that connects to the ER on the outside and to the nuclear lamina (and heterochromatin) on the inside.  The lamin B receptor (LBR) can be found in the nuclear envelope, as it is an integral membrane protein.  Also contained in the envelope is the nuclear pore complex.  The NPC is from 50-to-150 nm in size, with about 5,000 found on the membrane per nucleus.  At the NPC is where the inner and outer membranes of the nuclear envelope come together, and the NPC is involved in communication between chromatin and events outside the nucleus. 

The nuclear pore complex can pass 500 macromolecules per second, with molecules less than 5000 Da passing freely; those macromolecules larger than 60 kDa barely enter.  The channel that allows passage of these molecules is only approximately 10 nm wide.  The complex itself is made of 1000 proteins called nucleoporins, forming an octomeric structure.  It functions to import nuclear proteins via nuclear localization sequences (NLSs), a protein sequence that signals nuclear import.  A protein with more NLSs is imported more frequently, though an NLS does not mediate retention of the protein.  The protein importin, made of α and β subunits, assists in protein import by binding the NLS.  The importin receptor recognizes the NLS and then migrates on FG repeats of the nucleoporins to dock and translocate through the pore.  The cargo (with its NLS) is then released to the nucleus. 
Export of mRNA and proteins from the nucleus is also important to the cell.  It has been discovered that the sequence of the RNA does not affect its ability to export from the nucleus where it originated.  However, it has also been discovered that different RNAs are exported via different pathways, which may be facilitated by RNA binding proteins that contain a nuclear export sequence (NES).  Exports are the receptors for these NESs and facilitate protein movement out of the nucleus.  Those proteins with both an NES and NLS are considered shuttle proteins. 

Nuclear import and export is heavily regulated in the cell.  Phosphorylation can affect nuclear import: direct phosphorylation o the NLS can inhibit transport.  Ran-GTP is another important factor that affects transport.  Ran-GTP in the nucleus binds to empty receptors and transport them to the cytoplasm, where they can reload with a piece of cargo.  Ran-GAPs in the cytoplasm facilitate the hydrolysis of GTP to GDP, which promotes translocation of Ran-GDP to the nucleus.  Once in the nucleus, Ran-GEFs promote the exchange of GDP for GTP.  The interaction of the different forms of Ran allow for the recycling of shuttling proteins, allowing them to move proteins in and out of the nucleus.

The Nuclear Lamina
As mentioned, the nuclear envelope surrounds the nucleus and connects to the lamina on its inner face.  The nuclear lamina itself is about 75 nm thick and is composed of proteins similar to intermediate filaments, called lamins.  Lamins come in three forms: A, B, and C.  Lamins A and C bind heterochromatin.  Lamin B binds the lamin B receptor (LBR), which is connected to the nuclear envelope as an integral membrane protein.  The lamin complexes also peripherally bind to the NPC.  Lamins maintain the nucleus in its spherical shape, and phosphorylation of A and C subunits solubilizes them during prophase, allowing dissolution of the nuclear lamina.  Lamin B remains attached to its receptor during prophase, however.  Importantly, mutations in these proteins can cause laminopathies because they are involved in nuclear organization, and mutations can result in inhibited DNA synthesis.

The Inner Nuclear Matrix
Study of the inner nuclear matrix has shown that the protein composition is cell-type specific.  Additionally, chromosomes are not positioned randomly in the nucleus.  This has been exported by FISH using whole-chromosome probes.  It appears that chromosomes occupy specific territories and, at least in yeast, they take the Rabl conformation, with the telomeres and centromeres directly interacting with the nuclear lamina.  Matrix proteins function in both replication and transcription, and mRNAs from active genes can be found in the matrix, as can newly replicated DNA.  Additionally, matrix proteins of cancerous cells are different from normal cells.

Chromatin Review Articles:


Sunday, April 11, 2010

Oncogenesis Part 2: Genetic Instability, Colon Cancer, Transformation

Genetic Instability in Cancer Cells
A gene that receives a great deal of attention in cancer research is p53.  This protein acts as a tumor suppressor and is mutated in about 50% of all cancers.  Further, p53 is involved in a number of pathways, including apoptosis and genetic stability, so misregulation of the protein is a common factor in tumor cells.  Normally, very little p53 is present in cells, but it is induced during cellular stress.  When the cell experiences stress, p53 can induce apoptosis or cell cycle arrest by binding DNA and increasing p21 transcription, which acts as a CKI (see mitosis posts).  If p53 is lost, as it is in many cancers, the cell will replicate when it is not supposed to, and DNA accumulates a number of mutations (genetic instability).  Additionally, most cells will stop dividing when the telomeres shorten to a critical length, which is facilitated by p53.  When p53 is lost, even shortened telomeres don’t stop the cell from dividing, and genetic instability, again, is increased.  Some of these genetically unstable cells will upregulate telomerase, which will allow for continued proliferation. 

Colon Cancer Example
In normal colon cells, the APC protein inhibits cell cycle progression  by preventing Wnt from activating c-myc, which is required for progression from G1 to S.  If APC becomes mutated, the cell can progress through mitosis unchecked.  Because APC acts as a tumor suppressor, an individual must have two alleles that become mutated.  Individuals with a germline mutation in APC have an increased risk of colon cancer.  Further, if Ras becomes unregulated, it can stimulate MAPK signaling, leading to uncontrolled proliferation.

Colon cancer progresses through a number of stages:
  1. Normal epithelium
  2. Hyperplastic epithelium (via loss of APC)
  3. Early adenoma
  4. Intermediate adenoma (via activation of K-Ras)
  5. Late adenoma (via loss of Smad4 and other tumor suppressors)
  6. Carcinoma (via loss of p53)
  7. Metastasis

There exist a number of pathways that colon cells can become cancerous through the above stages.  The exact number of steps involved in malignant tumor progression is unknown, and the steps also vary based on type of tumor, though the general mechanism is similar.

Cell Senescence and Telomerase
In a study performed by Hayflick, cells that were explanted from tissue were shown to double roughly 60 times before entering senescence, a period when the telomeres are short, and the cells no longer proliferate.  Some cells are able to pass through the senescence stage and enter crisis, which lasts roughly 10-20 generations.  If a cell is able to pass through crisis and still undergo mitosis, it is considered immortal. 

Telomerase is the enzyme that can prevent cells from entering senescence.  When the catalytic subunit of telomerase (hTERT) is expressed, the telomeres are no longer degraded with each division.  With telomeres that are no longer shorted, there is no signal to p16INK4A through pRb and p53 to enter senescence, and the cell continues to divide.  The expression of hTERT in HEK (hamster embryonic kidney) cells prevents the entry of the cells into senescence.

Growth Signaling and Transformation
In order for a cell to divide, it must receive a number of signals that indicate that the environment is appropriate for it to divide.  In addition to dividing, tumor cells must be able to grow in size.  The growth signaling pathway that has received the most attention has been that involving Ras.  Nonetheless, there are a number of pathways that feed into cell proliferation signals, and these are often the genes that are altered in cancerous cells. 

In culture, cells that have been transformed exhibit the ability to form foci.  Cells in tissue culture are usually inhibited when a confluent monolayer has been established.  Those cells that are able to grow on top of each other in an unregulated fashion are considered transformed.  In a 3T3 cell, a common cell type used for understanding oncogenes, those cells that form a focus and are transformed have a mutation in p16, leading to a loss of function (p16 is a CKI)

The 3T3 Transformation Assay
  1. Transfect 3T3 cells with DNA from cancer cells
  2. Allow the cells to form foci
  3. Isolate DNA from foci and transform into new 3T3 cells
  4. Isolate DNA from new foci and generate a phage library
  5. Screen the phage library (with Alu probe) to identify human sequences

It is important to note that 3T3 cells are mouse cells, which facilitates the identification of human sequences using Alu elements (the mouse cells will not have these sequences).

Using the 3T3 transformation assay, the Ras onocogene was found.  The assay has also allowed for the identification of several other proto-oncogenes (genes that have the ability to become oncogenic).  Proto-oncogenes are usually activated via a gain-of-function mechanism, such as constitutive activity.  Approximately 100 oncogenes have been identified through the 3T3 assays and other methods.  Oncogenic collaboration is the cooperation between oncogenes to facilitate the faster formation of tumors. 

Proto-oncogenes can become oncogenes in several ways:
  1. Point mutations conferring constitutive activity
  2. Gene amplification leading to overexpression
  3. Chromosomal translocations putting the proto-oncogene under the control of a different promoter
  4. Chromosomal translocations that fuse two genes to make a chimeric protein with constitutive activity

Thursday, April 8, 2010

Oncogenesis Part 1: General Introduction

Oncogenesis
Benign tumors consist of cells that closely resemble and may function as normal cells that do not form malignant tumors.  They remain localized and stay small, with a fibrous capsule bounding the tumor.  However, if the benign tumor begins to interfere with normal function of other cells, such as via secretion of substances (such as hormones), the tumor can become a problem. 

In the case of malignant tumors, the cells express some characteristic proteins, but they grow out of control and more rapidly.  Malignant tumors also invade other tissues and can grow in sites vastly different from where they originated in a process terms metastasis.  The most diagnostic characteristic of a malignant tumor is its ability to invade other tissues.  These cells can break contacts with other cells and pass through the basal lamina to reach different areas of the body.  To facilitate this, cancer cells can secrete proteases such as plasminogen activator.  Plasminogen activator results in the formation of active plasmin from plasminogen, which helps to break down the basal lamina.  When the cell has passed the basal lamina, it can enter the blood stream and move to nearly any site in the body.  Roughly on in a million cells will be able to colonize another tissue, a hallmark of malignant tumors. 

Malignant tumor cells also have a high nucleus-to-cytoplasm ratio, with prominent nucleoli and many mitochondria, indicating high metabolic rates and significant growth.  Additionally, these cells appear to be de-differentiated, with few specialized structures that we would normally see in a cell.  Based on the cell’s gene expression and morphology, one usually is able to identify the source of a malignant tumor because it does retain some of the characteristics of its cellular origin. 

Carcinomas are cancers that have derived from the endoderm or ectoderm, while sarcomes are derived from the mesoderm.  Approximately 200 different cancer types have been identified, and there are approximately 300 different cell types in the body, meaning that cancer can arise from nearly any cell.

When a benign tumor grows, it is largely limited in size due to the inability for the tumor to acquire nutrients.  These tumors rely on diffusion to fuel the tumor cells.  In contrast, malignant tumors grow large, and in order to provide the nutrients necessary for growth of the tumor, they must recruit the formation of blood vessels in a process termed angiogenesis.  When a malignant tumor reaches a size over one million cells (or about 2 mm in diameter), it will induce the formation of blood vessels to provide nutrients to more cells.  Factors such as bFGF, TGFα, and VEGF are secreted by many tumors to facilitate angiogenesis.  Malignant tumor cells can also secrete factors that affect nearby cells to promote angiogenesis.  The larger the primary tumor, the higher the risk of metastasis is. 
Steps in metastasis of epithelial cells
  1. Upregulated cell growth in epithelium
  2. Invasion of the basal lamina via an invadopodium
  3. Entry into blood vessels; traveling through  the bloodstream
  4. Adherence to blood vessel wall
  5. Escape from blood vessel
  6. Colonizing of foreign tissue

The invadopodium consists of all the factors one would expect in an appendage to the cell: actin regulators such as WASP and Arp2/3; signaling molecules such as Cdc42; adhesion molecules such as integrins; and membrane remodeling complexes.

Tumors can be viewed as complex tissues in which the cell types have mutated to make a new phenotype (neoplastic phenotype).  It is important to note that these tumors are not growing in isolation, and they require interactions with non-cancer cells to grow as well.  The newest cancer therapies have begun to target these interactions in hopes of stemming tumor growth.

Genetically, cancer cells are messed up.  Therefore, one mutation in a cell does not cause cancer:  there are many different genes and factors that must become differently active for cancer to evolve.  Many different types of cancers evolve over time, meaning that the longer one lives, the higher the chance of cancer developing.  The multi-hit model indicates that successive mutations in a cell, each of which confers a growth advantage, will promote cancer development. 

For cancer cells to survive and proliferate unchecked, there are a number of functional capabilities that it must alter:
  1. Self-sufficiency in growth signals
  2. Insensitivity to anti-growth signals
  3. Tissue invasion (metastasis)
  4. Limitless replication
  5. Angiogenesis
  6. Evasion of apoptosis

There are a number of ways for a cancer cell to alter these pathways, and the order in which these capabilities emerge is different based on cell type and cancer type.  Additionally, there are a number of ways that cells are able to alter their function.  For example, the mutation of pRb can allow the cell to become immortalized and resist growth inhibition.  Mutations in p53 confer apoptosis evasion, resistance to growth invasion, and immortalization.  hTERT, which is the catalytic domain of telomerase, mutations will allow unlimited replication.  Mutations in Ras will allow for apoptotic evasion, growth in the absence of signals, angiogenesis, and metastasis.  Finally, mutations in PP2A (protein phosphatase 2A) affects signaling pathways that can affect a number of these alterations in cellular function.  

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.  

Friday, April 2, 2010

Chromatin Chapter 2: Modifications and the Histone Code

As mentioned, histones can be modified as well (this was mentioned briefly in the DNA repair posts).  Poly(ADP)-ribosylation causes the histones to bind less tightly to the DNA via repulsive negative charges.  This modification can be found at sites of DNA under repair.  Ubiquitination of H2A and H2B marks the proteins for degradation.  Phosphorylation tends to compact chromatin, but the mechanism by which this is done is unknown.  Finally, acetylation via HATs (histone acetyl transferases) on lysine and arginine residues in the core histones neutralizes the positive charge of the histones and causes them to repel from the DNA, leading to enhanced transcriptional activity. 

Modification
Effect
Poly(ADP) Ribosylation
Histones repel DNA, opening it for DNA repair
Ubiquitination
Degradation of H2A and H2B
Phosphorylation
Chromatin compaction
Acetylation
Histones repel DNA, opening it for transcription and DNA repair

Naively, one would consider the most important aspect of gene regulation to be the primary DNA sequence.  Nonetheless, chromatin plays a part in both transcription and replication, and the importance of this regulation is becoming more and more apparent.  One of the primary mechanisms by which replication and transcription are regulated via chromatin is from acetylation of histones.  Acetylation is performed by histone acetyl transferases (HATs), of which there are two kinds: A-HATs that mediate transcription-related acetylation, and B-HATs that modulate replication-related acetylation.  The most frequently observed acetylation events occur on histones H3 and H4, with A-HAT acetylating lysines 8 and 16 of H4 and 14 of H3; B-HAT acetylates lysines 5 and 12 of H4 and 9 of H3.

During DNA replication (when B-HATs are most active); histone acetylation is linked to chromatin maturation of the nascent DNA strand.  p48 is a histone escort that is in complex with a B-HAT, (Hat1p).  Chromatin assembly factor (CAF1) displaces B-HAT to form the chromatin assembly complex.  Histone de-acetylase (HD1) displaces CAF1 in the complex and removes acetylation events to mature the chromatin.  Acetylation events that remain on histones are recognized by specific proteins with specific domains, called bromodomains, that recognize and bind the acetylated lysine residues.

In contrast to histone acetylation, methylation via histone methyl transferases (such as human SUV39H1) mediates gene repression.  Gene activity is modulated by the balance of methylation and acetylation.  As mentioned, there are proteins that bind acetylated histones via bromodomains; similarly, there are proteins with chromodomains that bind methylated histones.  Two such proteins are Swi6 and HP1 (heterochromatin protein 1). 

It is important to note that actively transcribed genes are complexed with nucleosomes.  However, the chromatin in the region of these active genes tend to have acetylated histones and are also tend to be missing a couple nucleosomes from the promoter region.  Histone H1, the linker histone, is also missing from regions of the gene, and little DNA methylation is detected.  The DNA at actively transcribed genes is also more susceptible to nuclease attack and digestion.   

The Histone Code
Data considering differential gene regulation based on histone methylation and acetylation lead to the development of the histone code hypothesis.  The histone code is considered the pattern of markings on histones that cover the DNA.  Specific markings of these histones indicate newly-replicated chromatin, damaged DNA, and transcriptionally active or repressed regions.  Reader complexes recognize the marks on the chromatin and perform a function, while writer complexes such as HATs and methyltransferases mark the nucleosomes.  Write-read cycles spread chromatinization along DNA by consisting of DNA reader/writer complexes that bind a modified histone and create a marking on a nearby histone that then propagates chromatin modifications.  Barrier DNA sequences block these reader-writer complexes from expanding beyond a set boundary.   
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