C2006/F2402 '11 OUTLINE OF LECTURE #8

(c) 2011 Dr. Deborah Mowshowitz, Columbia University, New York, NY.

Last update 02/15/2011 02:42 PM

Handouts:  8A -- Heterochromatin vs Euchromatin; Basic Nucleosome Structure
                  8B -- Details of Nucleosome & chromatin structure. Similar to Becker figs. 18-20 & 18-21.
                            For color versions of nucleosome pictures on 8B click here.
                  8C -- Testing the state of chromatin by resistance to degradation with DNase.

These handouts are posted on Courseworks since they contain copyrighted material. Extra copies of all paper handouts are in boxes on 7th floor of Fairchild.

I. Nuclear Structure & Function, cont: Nucleoli & RNA polymerases

    A. Nucleolus

1. Nucleolus has no membrane (see Becker fig. 18-33). 

2. Nucleolus is site of rRNA synthesis and assembly of ribosomal subunits

a. Role of Nuclear Pores -- Production of cytoplasmic ribosomes requires extensive traffic in and out of nucleus

• All transport in and out of nucleus is through nuclear pores.

• Both RNA and protein (& completed subunits) must pass through pores.

• Reminder: importin goes into the nucleus with the 'cargo' protein it is transporting in; exportin goes out with its cargo; both proteins then recycle. (See Becker 18-30.)

• Synthesis of ribosomal subunits of cytoplasmic ribosomes requires traffic in both directions -- see below.

b. Synthesis of rRNA & protein

(1). rRNA for cytoplasmic ribosomes is made in the nucleolus (see Becker fig. 18-34) using RNA polymerase I (see below)

(2). Ribosomal proteins are NOT made in the nucleolus. Ribo proteins are made in the cytoplasm, but imported into the nucleus for assembly into subunits.

• Messenger RNA for ribosomal proteins is made in the nucleus using polymerase II (see below) and exported to the cytoplasm, as for any other mRNA.

• Messenger RNA for ribosomal proteins is translated in the cytoplasm.

• After the mRNA is translated, the proteins are imported back into the nucleus for assembly with rRNA.

c. Assembly of subunits is in the nucleolus Ribosomal subunits destined for the cytosol are assembled in the nucleolus from ribosomal proteins (made in cytoplasm) and rRNA. 

d. Function of ribosomes is in the cytoplasm -- Completed ribosomal subunits are shipped out to the cytoplasm (through nuclear pores) to be used in protein synthesis. **

**Note: Becker says there are ribosomes translating mRNA in the nucleus. Maybe, maybe not. This finding is controversial -- the existence of nuclear ribosomes is not firmly established, so we are ignoring them. Ribo proteins (for both cytoplasmic and mitochondrial ribosomes) are made in the cytoplasm in any case.

4. Nucleolus vs NOR (= nucleolar organizer region of DNA)

a.  Number of rRNA genes --There are multiple copies of the genes for rRNA; many copies are located in tandem. There may be one or multiple clusters of rRNA genes. Each cluster = one NOR (see below).

b. Why many copies of rRNA genes?  Multiple copies are needed because rRNA is made directly off the DNA -- no intermediate such as mRNA to allow for amplification.

Q to think about: Do you need multiple copies of the genes for tRNA?

c. NOR =  region of the DNA coding for rRNA = rDNA = genes for rRNA.

• Each NOR has multiple copies of the rRNA genes.

• Genome may have one or more NORs.

• NORs are permanent -- are part of DNA.

d. Nucleolus  = structure seen in interphase; not in meiosis/mitosis.

• Nucleolus forms during interphase at location of one or more NORs.

• Several NORs can cluster together to form one nucleolus.

• Nucleoli are only visible when NOR is being transcribed and ribosomes are being assembled.

e. What can you see? -- Nucleolus  (but not NOR) disappears during meiosis/mitosis since genes for rRNA inactive then (no rRNA made or ribosomal subunits assembled)

        5. Organelle ribosomes --  made and assembled differently from cytoplasmic ribosomes

a. rRNA -- rRNA for mitochondrial ribosomes is encoded in mito DNA & made inside the mitochondria

b. proteins -- the proteins for mitochondrial ribosomes are encoded in the nucleus and made in the cytoplasm.

    B. RNA polymerases & associated proteins

1. In Prokaryotes -- there is only one RNA polymerase.

2. In eukaryotes -- there are three RNA polymerases. Differ in sensitivity to drugs, location in nucleus, & what RNA's they make. (See Becker table. 21-1 for details; for reference only.)

a. RNA pol I → big ribosomal RNAs. (All ribo RNA but 5S.) This is the polymerase found in the nucleolus. 

b. RNA pol II → mRNA (We'll focus primarily on this one when we discuss regulation of transcription.)

c. RNA pol III → small RNA's like tRNA, 5S RNA (small ribosomal RNA)

3. Eukaryotic Transcription factors (TFs)

        a. Eukaryotic transcription requires additional proteins (TFs), not just RNA polymerase.

        b. Binding -- RNA Polymerase will not bind to DNA directly -- binding requires TFs.

        c. TFs are usually divided into two classes (more details next time):

(1). basal (or general) -- required for transcription of all genes; are the same in all cells

(2). regulatory (or tissue specific) -- decrease or increase basal transcription (usually called repressors or activators respectively). Are different in different cell types.

To review nuclear structure & transport into nuclei, try problems 4-2, 4-6 parts A & B,  and 4-7, parts A & C.

    C. Chromatin. DNA in eukaryotes is not "naked" as it is in bacteria. It is in the form of chromatin = DNA complexed with histone proteins. So how do polymerases and TFs get to the DNA and how do they manage to carry out  transcription with all those proteins stuck to the DNA? What actually turns transcription on and off? To answer these questions, we need to look first at chromatin structure.  
 

II. General Chromatin Structure -- Euchromatin & Heterochromatin   

    A. Composition of chromatin = DNA + associated proteins

1. Associated proteins are mostly histones (small, basic; more details below)

2. Associated proteins includes some (nonhistone) regulatory proteins

    B. States of chromatin

1. In all states, DNA has proteins attached

2. Usually differences are due to different states of folding after histones added, not removal of histones (there are some exceptions which will be discussed later).

3. Two Types of Chromatin -- Two basic states of chromatin are visible in the light microscope

a. Heterochromatin or heterochromatic chromatin/DNA

(1). What is it? Chromatin that is darkly stained, & relatively condensed (tighter),

(2). Heterochromatin is genetically inactive (not transcribed)

(3). Two kinds of (interphase) heterochromatin

(a). Constitutive heterochromatin 

• Always heterochromatic in interphase in all cells

• Example: chromatin at centromeres, telomeres

• Usually repeating in sequence, non coding.

(b). Facultative heterochromatin

• Sometimes heterochromatic in interphase, sometimes not (depends on tissue, time etc.).

• Example: inactive X. Same X is not inactive in all cells.

• Whichever X is inactive (forms a Barr body) is heterochromatic.

(4). Is all inactive chromatin heterochromatic? No. Most of the DNA that is inactive during interphase (in a particular cell type) is euchromatic, NOT heterochromatic.

(5). Mitotic chromatin -- All chromatin is heterochromatic (tightly condensed) during mitosis & meiosis.

(6). All chromatin, both heterochromatin & euchromatin is replicated in S. Heterochromatic DNA is not genetically active (not transcribed), but it is replicated.

b. Euchromatin or euchromatic chromatin/DNA

(1). What is it? Chromatin that stains more lightly, is less condensed (looser).

(2). Important Features of Euchromatin

• Capable of genetic activity (transcription) as vs. heterochromatin.

• Normal state of most DNA during interphase.  

• Transcribable, but not necessarily being transcribed now. DNA must be euchromatic to be active, but not all euchromatin is active. 

(3). Reminder: Most interphase DNA is euchromatic, whether it is transcribed or not.

(4). Not all euchromatin is folded equally tightly

(a). Tighter vs looser. Euchromatin is often divided into several distinct states of folding,  although tightness of folding is probably really continuous from relatively loose ↔ relatively tight.

(b). Different states of (eu)chromatin look about the same in the light microscope.  Therefore indirect methods (such as DNase treatment) are necessary to test state of folding.

(c). Correlation between folding and function. In general, more active (transcribed) chromatin is looser, but the situation is complex .

III. Structure of Chromatin -- Histones & Nucleosomes

    B. Model for Chromatin (Beads on string level) -- nucleosomes (See handout 8A and Becker fig. 18-20 or Sadava fig. 11.9 (9.8). Where is the protein relative to the DNA?

1. Octamer of 2 each of H2A, H2B, H3, H4 (+ some DNA) = one bead.

2. DNA wound 2X around (on outside of) each octamer -- protects core. For more pictures and info on nucleosomes, click here.

3. Linker DNA between cores = 50-60 BP; most exposed & sensitive to some nucleases (relative to DNA in core).

4. H1 is on outside of DNA/octamer (See handout 8A or click here for model)

5. Nucleosome = repeating unit = 200 BP DNA + octamer (H1 optional).

    C. Nucleosomes & Higher Levels of Structure (requires H1) -- how does chromatin fold up? See 8B or books for detailed pictures. (Becker fig. 18-21 or Sadava fig. 11.9 (9.8) or click here & scroll down.  Stages of folding are as follows (details in table below):

a. Chromatids: Folds back on self to form structures/fibers about 700 nm across (per chromatid)

b. At metaphase = tightest = 1/15,000-1/20,000 orig. length (Chromosome is 4-5 microns long but each chromatid contains about 75 mm of double helical DNA)

IV. Does Folding of Chromatin Correlate with Genetic Activity?

A. Basic Issue/Question: In interphase, almost all chromatin is euchromatic, but not all euchromatin is transcribed in any one cell.  Is all euchromatin folded to the same degree or not? Does looseness or tightness of folding correlate with level of genetic activity?

B. How do you test state of euchromatin?

1. Why use DNase?

a. All (eu)chromatin looks about the same in the light microscope.  Therefore indirect methods (such as DNase treatment) are necessary to test state of folding.

b. Use of DNases.  States of folding of (eu)chromatin are often distinguished by effects of treatment of chromatin (not DNA) with various types of DNase.

(1). Rationale: DNA that is in tighter areas of chromatin will be more protected from degradation. (Some examples of this are discussed below and are in problem sets.)  

(2). Results: State of (eu)chromatin can be deduced from relative sensitivity of DNA (while still in chromatin) to degradation by various DNases.

2. Basic Method -- see Becker fig. 23-17 or handout 8C. Want to examine chromatin region containing a particular gene, say globin genes, from dif. tissues.

a. Issue -- This region is euchromatic in interphase in all these tissues, even though it is not expressed in all of them. No gross difference is visible, but is there a difference in folding?

b. What you want to do: Fish out section of chromatin containing globin genes (from cells that make globin and cells that don't) and test state of chromatin folding.

c. What you have to do: Need to test for folding indirectly, so you need to do experiment in reverse. You distinguish states of folding of chromatin first (indirectly) and then find which state contained the genes of interest, say, globin genes.

        3. Actual Experimental Procedure

a. Overall:

(1). Treat chromatin with DNase, differentially degrading DNA in active and inactive chromatin.

(2). Remove protein to leave the DNA.

(3). Examine DNA -- Use probe to test DNA for state of  genes of interest and see if the genes were degraded or not. 

b. Details of DNase treatment: (step 1 on handout 8B)

(1). Treat chromatin -- not DNA -- with DNase. Then isolate DNA and see what state it's in.

(2). DNase used is not micrococcal nuclease.

(a). Usually use DNase I, a different enzyme from one used previously to get a "ladder". 

(b). DNase I cuts the DNA differently than micrococcal nuclease.

• DNA that is simply wound around nucleosomes is not totally protected from DNase I -- further folding up is required. So resistance to DNase I is a measure of how tightly the nucleosomes are folded in on themselves.
   

• DNase I does not cut preferentially at any particular sequence or at any particular place relative to the position of the nucleosome. Therefore it does not give a "ladder." 

(3). Can vary conditions -- amount of enzyme, time, etc. to distinguish various degrees of sensitivity to the enzyme. 

(4). Can test chromatin from many different tissues, say erythrocytes (in chickens -- still have nuclei) vs. brain

(5). Can test state of many different genes using diff. probes (see below).

c.  Expected result:

(1). If euchromatin is relatively loose, DNA will be unprotected and readily degraded by DNase I. There will be nothing left to hybridize to the probe for, say, globin.

(2). If euchromatin is relatively tight, DNA in that region will NOT be easily degraded, and there will be DNA left to hybridize to probe.

d. How to measure state of DNA (after treatment of chromatin with DNase I). Cut purified DNA with restriction enzyme and do a Southern blot with a probe to the gene of interest.  Details:

(1). Prepare DNA.

    Step 2A on handout: Extract DNA (remove histones) -- get naked DNA.

    Step 2B: Treat naked DNA with restriction enzymes -- to get pieces of reasonable size

• Will give discrete pieces IF DNA was protected from DNase I (in relatively tight region of chromatin)

• If DNA was in loose region of chromatin, DNA will already have been degraded by DNase I.  

(2). Find regions corresponding to known genes. Do Southern -- steps 2B to 3B.

    Step 3A.  Run restriction fragments on gel, blot to solid support.

    Step 3B. Hybridize to labeled probe to identify DNA from regions of interest (say, globin genes).

    Result: Only DNA from areas with relatively tight chromatin will give clear, undegraded (labeled) bands on the gel. (See figures on handout.)

To review how this is done, try problem 4-9. Also ask yourself, suppose you do the experiment on the handout, but you use a probe to either (1) RNA polymerase II gene or (2) gene for type IV IF (IF of neurons). What would the blots look like?

3. Summary of results of treatment of euchromatin with DNase I and other nucleases.

a. Almost all eukaryotic DNA is associated with some protein. How do we know? -- Almost all DNA is much more resistant to digestion by DNase I than naked DNA

b. Almost all eukaryotic DNA is in nucleosomes. All DNA but the very loosest euchromatin (see d) is in nucleosomes, and gives a ladder when treated with microccal nuclease. 

c. Not all euchromatin is equally loose (equally sensitive to DNase), but all euchromatin is packed less tightly than heterochromatin.  See table on handout 8A.

d. Actually transcribed (coding) DNA is more sensitive to DNase I than ordinary euchromatic DNA.

(1). Transcribed chromatin is 'looser' but it is still in nucleosomes -- it is much more resistant to DNase than naked DNA.

 (2). Nucleosomes are not stripped off during genetic activity. One or two nucleosomes, at the most, are probably moved aside as the polymerase moves relative to DNA during transcription or DNA replication.

(3). 'Looser' euchromatin may correspond to stretched out loops that have been unfolded to the 10nm fiber or beads-on-a-string stage. Includes genes that are being actively transcribed, or were transcribed recently, or are next to transcribed regions, etc.

e. Hypersensitive sites exist -- these are the only sections of euchromatin that are not in nucleosomes.* See Becker fig. 23-18. These are the 'loosest' regions of euchromatin.

(1). Some hypersensitive sites found = very sensitive regions (100X more sensitive to degradation by DNase I than heterochromatin; 10X more sensitive than active euchromatin.) Degraded by very low amounts of DNase I.

(2). Hypersensitive sites correspond to regulatory, not coding, regions (in areas of active transcription).

(3). Hypersensitive sites are different in different tissues -- different genes are 'turned on' (transcribed) in each cell type. DNA is the same in (almost) all cells, but only the genes that are 'turned on' (in that cell type) will have hypersensitive sites.

(3). Hypersensitive sites correspond to sites without nucleosomes (or to regions with very loose nucleosomes -- see 5b).

(4). DNA in hypersensitive sites is not naked -- it is bound to regulatory proteins.

(5). Why are hypersensitive sites so sensitive to DNase? *

a. The traditional view: Transcription factors (regulatory proteins) have replaced histones and the other proteins don't protect the DNA as well as histones do. See Becker fig. 23-18.

b. An alternative view: Histones are still present, at least in some hypersensitive sites, in addition to regulatory proteins. The histones  are much more loosely attached to DNA than in normal nucleosomes, so the DNA is not as well protected as in ordinary nucleosomes.

* When doing the problems, assume that hypersensitive sites have no nucleosomes, unless the data in a particular experiment indicates otherwise.

To review the differences in states of chromatin, uses of DNase, etc., try the rest of 4-3, 4-9 & 4-10.

V. How Do you turn a Eukaryotic Gene On?

    A. The Problem: Need to unfold/loosen chromatin before transcription is possible. Can't just add RNA polymerase (& basal TFs) to DNA and start transcription. DNA is in chromatin and must be made accessible.

    B. So how can transcription occur?  This will be the subject of the next lecture.