C2006/F2402 '05 OUTLINE OF LECTURE #9
(c) 2005 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 02/21/2005 10:40 AM (A few extra references to texts added; rest is same.)
Handouts (not on web): 9A & 9B (nucleosome & chromatin structure) & 9C (Article from Times on Nobel Prize '04.)
I. Nuclear Structure and Role of Nucleolus -- see notes for Lecture #8.
II. General Chromatin Structure -- Euchromatin & Heterochromatin
A. The Issue: DNA in eukaryotes is not "naked" as it is in bacteria. It is in the form of chromatin = DNA complexed with proteins. So how do polymerases and regulatory proteins (generally called transcription factors or TFs in eukaryotes) get to the DNA and how do they manage to carry out (& regulate) transcription with all those proteins stuck to the DNA? To answer these questions, we need to look first at chromatin structure.
B. 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
C. 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 Basic States of Chromatin (visible in the light microscope)
(1). Darkly stained, relatively condensed, genetically inactive
(2). Two kinds of (interphase) heterochromatin
(a). Constitutive heterochromatin -- always heterochromatic (ex: 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.
(3). All DNA (chromatin) is heterochromatic during mitosis.
(1). Stains more lightly, less condensed.
(2) Capable of genetic activity (transcription) as vs. heterochromatin. Normal state of most DNA during interphase. (Most interphase DNA is euchromatic, whether it is transcribed or not.) DNA must be euchromatic to be active, but not all euchromatin is active.
(3). 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). Use of DNases. States of folding of (eu)chromatin are often distinguished by effects of treatment with various types of DNase. State of (eu)chromatin will determine relative sensitivity of DNA (while still in chromatin) to degradation by various DNases. DNA that is in tighter areas of chromatin will be more protected from degradation. (Some examples of this will be discussed below and are in problem sets.)
(d). Correlation between folding and function. In general, more active (transcribed) chromatin is looser, but the situation is complex -- see details below.
III. Histones & Details of Chromatin Structure
A. Structure of Chromatin -- how are proteins and DNA arranged? The evidence.
1. Appearance in EM -- low salt ("beads on a string" appearance) vs physiological salt (30 nm fiber). See Becker fig. 16-17 or Purves fig. 9.6 (9.7).
2. Results of treatment with DNase (microccocal nuclease; cuts exposed DNA at random; does not cut at specific base sequences.)
a. Treat chromatin with a little mic. nuclease; then remove protein and isolate the DNA, then run DNA on a gel. Get 200 Base Pair (BP) "ladder" = sequences of multiples of 200 BP on gel. Implies repeating structure ("bead") with about 200 BP per repeat; DNA exposed (so easily cut) about once per 200 BP. (See Becker fig. 16-18 & 16-19.)
b. More treatment with nuclease --> resistant core of 140-145 BP. Implies part of 200 BP repeat is relatively protected in/on core of "bead"; rest goes between beads and is more exposed.
a. 5 types: H2A, H2B (slightly lys rich), H3, H4 (arg rich) & H1 (lys rich). All relatively small, basic proteins.
b. All histones are highly conserved evolutionarily (this implies histones carry out a critical function that depends on a particular structure -- can't change structure much without ruining function).
b. How much per 200 BP of DNA? 2 molecules each of H2A, H2B, H3, H4 plus one molecule of H1.
c. Low salt removes H1.
B. Model for Chromatin (Beads on string level) -- nucleosomes (See small picture in '91 article (9A) and Becker fig. 16-20 or Purves fig. 9.6 (9.7)). 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.
3. Linker DNA between cores = 50-60 BP; most exposed & most sensitive to nuclease used above.
4. H1 is on outside of DNA/octamer
5. Nucleosome = repeating unit = 200 BP DNA + octamer (H1 optional).
C. Nucleosomes & Higher Levels of Structure (requires H1) -- how does chromatin fold up? See big picture in '97 article (9B) -- and books for detailed pictures. (Becker fig. 16-21 or Purves fig. 9.6 (9.7)) Stages of folding are as follows (details in table):
1. Nucleosomes. DNA + histones form a chain of nucleosomes; about 1/7 original length of DNA (see table below).
2. 30 nm fiber. Chain of nucleosomes folds back on itself (supercoils) forming 30 nm fiber (sometimes called solenoid). Exact structure unclear. Fiber is about 3 beads across; 6 beads/turn = 1/42 length of DNA. Need tails (of histones) and H1 to form 30nm fiber. (See 97 article.)
3. Loops. 30 nm fiber forms loops about 300nm in diameter (1/750 orig. length). Different sections may be tighter or looser. Individual loops are stretched out (probably to beads-on-a-string stage) when actually transcribed.
4. Higher Orders of Folding. Looped structure folds further --> --> heterochromatin (not transcribable)
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 contains 75 mm of double helical DNA)
D. Summary of States of Folding -- compare to handout 9B.
Compaction relative to previous
Nucleosomes -- beads on a string
7 (1/7th length of DNA)
30 nm fiber
700 nm (per chromatid)
* Packing ratio = length of DNA or DNA/protein complex relative to original length of DNA.
E. Modifications of histones -- helps tighten up or loosen chromatin
1. Histones have tails that stick out (see handout 9B).
a. Many different modifications of amino acids in tail are possible: For example, addition/removal of acetate, methyl or phosphate groups to side chains of AA.
b. Regulatory Function: Modifications of histones affect folding of chromatin and therefore activity of genes.
2. Some Examples:
a. Phosphorylation of H1 occurs in M; changes in kinase and phosphatase activity affect state of histones and folding of chromatin in parallel with changes in lamins.
b. Acetylation of lys side chains of histones. Acetylation of histones --> more active, looser chromatin (see articles). Acetylation of H3 & H4 is higher in active chromatin.
c. Other modifications, such as methylation, occur and are thought to serve regulatory purposes as well. (Both DNA and histones can be methylated.)
To review nucleosome structure, try problems 4-1, 4-3 A, 4-4 A & 4-8 A.
IV. Does Genetic Activity Correlates with States of Folding of Euk. Chromatin? How to test?
A. Polytene chromosomes (See Becker Ch. 21 esp. fig. 21-15 & 21-16) Special Interesting Case. Will not be covered in lecture; is here FYI.
a. Special case where many chromatids -- about 1000 -- are lined up together with genes and loops of chromatin aligned; therefore can see differences in state of folding in light microscope.
b. Can compare changes in folding of chromatin and level of transcription (by incorporation of labeled U) in a single tissue as it responds to hormones that alter gene expression.
2. Disadvantage: Can't compare regions of active/inactive chromatin from many dif. tissues.
3. Results: Active (transcribed) regions are clearly looser -- individual loops stretched out more. (See Becker )
B. Results of treatment with DNase I (of ordinary chromosomes/chromatin) -- How folded is Euchromatin?
1. The problem -- Not all euchromatin is transcribed in any one cell -- is it all folded to the same degree or not?
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 with various types of DNase. State of (eu)chromatin will determine relative sensitivity of DNA (while still in chromatin) to degradation by various DNases. DNA that is in tighter areas of chromatin will be more protected from degradation. (Some examples of this will be discussed below and are in problem sets.)
2. Method -- see Becker fig. 21-17. Want to examine chromatin region containing a particular gene, say globin genes, from dif. tissues. (Note: 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; that's why indirect methods are necessary.)
a. 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.
b. What you have to do: Need to test for folding indirectly, and to do experiment in reverse. Test state of folding first (indirectly) and then test state of genes.
c. General procedure: Treat chromatin with DNase, differentially degrading DNA in active and inactive chromatin. Then use probe to test for state of known genes and see if genes were degraded or not.
d. Details of DNase treatment: Treat chromatin with DNase (Usually DNase I, a different enzyme from one used previously to get a "ladder"). Then isolate DNA and see what state it's in.
(1). Can vary conditions -- amount of enzyme, time, etc. to distinguish various degrees of sensitivity to the enzyme.
(2). Can test chromatin from many different tissues, say erythrocytes (in chickens -- still have nuclei) vs. brain
(3). Can test state of many different genes using diff. probes (in step e below).
(4). 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. Does not give "ladder."
e. Expected result: If chromatin 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.) If chromatin is relatively tight, DNA in that region will NOT be easily degraded. (And there will be DNA to hybridize to probe.)
f. Details of how to measure state of DNA (after treatment of chromatin with DNase I):
(1). Prepare DNA. Extract DNA (remove histones); treat naked DNA with restriction enzyme (to give pieces of reasonable size IF DNA was protected from DNase I). If DNA was in loose region of chromatin, it will already have been degraded by DNase I.
(2). Find regions corresponding to known genes. Run restriction fragments on gel; do blot, identify regions of interest (say, globin genes) with probe. Only regions from areas with relatively tight chromatin will give clear, undegraded bands on the gel.
3. Results of treatment of euchromatin with DNase I and other nucleases.
a. Almost all eukaryotic DNA is in nucleosomes. (See (c) for exceptions.) How do we know?
(1). Almost all DNA is much more resistant to digestion by DNase I than naked DNA
(2). Almost all DNA forms ladder when treated with micrococcal DNase.
b. Actually transcribed (coding) DNA is more sensitive to DNase I than ordinary euchromatic DNA. But transcribed DNA is much more resistant than naked DNA -- it is still in nucleosomes.
c. Hypersensitive sites exist -- these are the only sections not in nucleosomes.
(1). Some hypersensitive sites found = very sensitive regions (100X more sensitive to degradation by DNase I than heterochromatin; 10X more sensitive than active euchromatin.)
(2). Hypersensitive sites correspond to sites without nucleosomes
(3). DNA in hypersensitive sites is not naked -- it has other proteins, but no histones. See Becker fig. 21-18.
(4). Hypersensitive sites correspond to regulatory, not coding, regions (in areas of active transcription).
(5). Why are hypersensitive sites so sensitive to DNase? Transcription factors (regulatory proteins) have replaced histones and the other proteins don't protect the DNA as well as histones do.
C. Possible States of Interphase Chromatin -- what is inferred from all the results, and the implications. (See table on handout. )
a. Super Tight = heterochromatin = essentially not uncoiled from mitosis. Genetically inactive.
b. Euchromatin -- 3 major states?
(1). Loose. Looser than heterochromatin, but not being transcribed (genetically inactive) at the moment. 30 nm fiber?
(2). Looser -- but still has nucleosomes. Beads-on-a-string? Stretched out loops? Example: coding region that are active -- they are actually being transcribed. (Also includes genes that were transcribed recently, or are next to transcribed regions, etc.)
(3). Loosest or hypersensitive region (missing some histones; has regulatory proteins instead). Example: active promotor or enhancer.
c. A caution: There are probably intermediate states, and the ones above are simply the only ones that have been clearly distinguished using the methods currently available. For example, all inactive euchromatin is probably not the same. See ** below.
To review the differences in states of chromatin, uses of DNase, etc., try the rest of 4-3 to 4-8.
V. Introduction to Regulation of Eukaryotic Gene Expression
A. What has to be done to turn a eukaryotic gene on/off? What steps can be regulated?
1. In prokaryote (for comparison) -- process relatively simple.
a. Most regulation at transcription.
b. Translation in same compartment as transcription; translation follows automatically.
c. Most mRNA has short half-life.
2. In eukaryote -- Most regulation is at transcription, but you have more steps & complications -- more additional points of regulation. See Becker fig. 21-11.
a. Need to unfold/loosen chromatin before transcription possible. We know that modification of histones, binding of certain non-histone proteins, and/or methylation of DNA is correlated with state of folding. Not clear what is primary cause and what is secondary effect of unfolding. Two possible models (most current data favors the first):
(1) Two step model for regulation. See Becker fig. 21-10.
(a). First must de-condense (loosen up) euchromatin to a transcribable state = loose (compared to heterochromatin and compared to inactive euchromatin). Pull out 30nm fiber to beads-on-a-string stage?
(b). Then add transcription factors (more or less as for prokaryotes) --> actual transcription
(1). Regions with transcription factors = nucleosomes removed = hypersensitive sites
(2). Regions being transcribed = nucleosomes are somehow "loosened up" or "remodeled" but not removed.
(c). What changes state of chromatin? (To tighten or loosen.)
(1). Remodeling proteins: these are responsible for moving and/or loosening up nucleosomes. See Purves 14.16 (14.17).
(2). Enzymes that modify histone tails. Changes in modification may have a direct effect and/or affect binding of regulatory proteins.
(2). One Step Model -- addition of TF's is primarily what opens up the chromatin -- don't need a separate remodeling step first. Alternatively, both unfolding or decondensation and addition of TF's occur simultaneously.
b. Transcript must be processed (capped, spliced, polyadenylated, etc.) -- any of these steps can be regulated, and there is more than one way to process most primary transcripts. (An example will be discussed next time.)
c. Transcription & translation occur in separate compartments.
(1). mRNA must be transported to cytoplasm.
(2). Translation can be regulated (independently of transcription) -- can control usage of mRNA, not just supply of mRNA. (An example will be discussed next time.) For any particular mRNA, can regulate 1 or both of following:
(a). rate of initiation of mRNA translation (how often ribosomes attach and start translation)
(b). rate of degradation of mRNA
IV. Major features of gene regulation in Eukaryotes
A. How can amount of protein be controlled? If cell makes more or less protein, which step(s) are regulated? Many possible points of regulation in eukaryotes. See Becker fig. 21-11, or Purves 14.11 (14.13), and list of steps above.
1. Most common point of regulation is at transcription (in both euk. & prok.) If you need more protein, usually make more mRNA.
2. Transcription is not the only step controlled, especially in euk. (Some examples will be discussed later.)
B. If cells make different proteins, how is that controlled? If two eukaryotic cells (from a multicellular organism) make different proteins, what is (usually) different between them?
1. Is DNA different? (No, except in immune system.)
2. Is state of chromatin usually different? (Ans: yes) How is this tested? Method & result described above. See figure 21-17 in Becker. What causes the difference in states of chromatin? Not clear what is cause and what is effect.
3. Is mRNA usually different? (Ans: yes). This means you can get tissue specific sequences from a cDNA library. (cDNA library = collection of all cDNA's from a particular cell type.) DNA from each cell type is the same; mRNA and therefore cDNA is not. See Becker fig. 21-20.
4. If mRNA's are different, why is that? Is the difference due entirely to differences in transcription?
a. Transcription is different in different cells. It could be that all cells transcribe all genes, but only some RNA's are exported to the cytoplasm and the remaining nuclear RNAs are degraded. This is not the case. Only selected genes are transcribed in each cell type, and RNA's from those genes are processed to make mRNA.
b. Splicing and processing of same primary transcripts can be different (in different cells or at different times). Different mRNA's (& therefore proteins) can be produced from the same transcript by alternative splicing and/or poly A addition.
To review possible steps in regulation, try problems 4-9 to 4-11.
Next time: Details of Eukaryotic Transcription