C2006/F2402 '10 OUTLINE OF LECTURE #8
(c) 2010 Dr. Deborah Mowshowitz, Columbia University, New York, NY.
Last update 02/15/2010 03:16 PM
Handouts: 8A -- Regulatory Elements & Picture of a typical Eukaryotic Gene (not on web).
8B -- Testing the state of chromatin by resistance to degradation with DNase.
You will also need 7A & 7B for nucleosome structure.
Reminder: Extra copies of all handouts are in boxes on 7th floor of Fairchild.
I. Histones & Nucleosomes
A. Structure of Chromatin -- how are proteins and DNA arranged? How does all that DNA fit into a tiny cell? The evidence -- Details of 1 & 2 in last lecture.
a. DNA is in Nucleosomes: Chromatin = DNA-protein complex with a repeating structure. One nucleosome = one repeat unit = 200 BP + associated proteins.
1. Appearance in EM -- Shows DNA and protein arranged in repeating structure of 'beads on a string'.
2. Results of treatment with DNase
b. Nucleosome core: About 145 PB of 200 BP repeat is relatively protected in/on core of "bead" -- rest is a 'linker' that goes between beads and is more exposed.
c. Linker: Linker DNA has one site every 200 BP that is relatively unprotected and readily cut by micrococcal nuclease.
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. (It's on outside of bead; more easily removed.)
B. Model for Chromatin (Beads on string level) -- nucleosomes (See handout 7A and Becker fig. 18-20 or Sadava fig. 9.8 (9.6)). 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 7A 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 7B or books for detailed pictures. (Becker fig. 18-21 or Sadava fig. 9.8 (9.6) or click here. ) Stages of folding are as follows (details in table below):
1. Nucleosomes. DNA + histones form a chain of nucleosomes; about 1/7 original length of DNA (see table below). Also called a "10 nm fiber."
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.
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 7B.
|Structure||Compaction relative to previous||Packing Ratio*||Diameter||H1 Needed?|
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. As packing ratio increases, chromatin fibers get shorter and wider.
E. Modifications of histones -- helps tighten up or loosen chromatin
1. Histones have tails that stick out (see handout 7B or click here)
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 amino acids.
b. Regulatory Function: Modifications of histones affect folding of chromatin and binding to regulatory proteins; therefore they affect the 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. (Should kinases help chromatin get looser or tighter?)
b. Acetylation of lys side chains of histones. Acetylation of histones → more active, looser chromatin. Acetylation of H3 & H4 is higher in active chromatin.
c. Methylation -- In most organisms, both DNA and histones can be methylated. (Methyl groups can be added to C's in DNA as well as side chains of AA.)
(1). Effects In DNA.
Usually, but not always, DNA methylation is higher in more inactive/condensed chromatin.
In some organisms, there is no methylation of DNA.
(2). Effects in histones. It depends which amino acids are methylated -- some modifications increase likelihood of transcription, and some decrease it.
d. Overall histone modification 'code' -- it is possible that each combination of modifications to the histone tails has a specific meaning. For a full explanation (fyi) see Alberts. (Or go to PubMed at http://www.ncbi.nlm.nih.gov/sites/entrez , click on books on upper right, and enter 'histone code' in the search term box. )
To review nucleosome structure, try problems 4-1, 4-3 A, 4-6 C, 4-9 A, 4-7 B, & 4-10 A.
II. Does Folding of Chromatin Correlate with Genetic Activity?
A. States of Interphase Chromatin -- (See table on handout 9A )
1. Super Tight = heterochromatin = essentially not fully uncoiled from mitosis. Genetically inactive.
2. Euchromatin -- 3 major states?
a. Loose. Looser than heterochromatin, but not being transcribed (genetically inactive) at the moment or recently. 30 nm fiber?
b. Looser -- but still has nucleosomes. 10 nm fiber or beads-on-a-string state. 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.)
c. Loosest or hypersensitive region (missing some histones; has regulatory proteins instead). Example: active promoter (Promoter = region around where transcription starts; it's where RNA polymerase and associated proteins bind. More on this below.)
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.
B. How do you test state of (eu)chromatin? How do we know what's 'tight' or 'loose'?
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. How? 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.) Therefore, state of (eu)chromatin can be deduced from relative sensitivity of DNA (while still in chromatin) to degradation by various DNases.
2. Method -- see Becker fig. 23-17 or handout 8B. 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, 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.
c. How you do it: Treat chromatin with DNase, differentially degrading DNA in active and inactive chromatin. Then use probe to test for state of genes of interest and see if the genes were degraded or not.
d. 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 (in step e below).
e. Expected result: 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.) If euchromatin is relatively tight, DNA in that region will NOT be easily degraded. (And there will be DNA to hybridize to probe.)
f. 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) DNA polymerase I 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. 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. One or two nucleosomes, at the most, are probably moved aside as the polymerase moves relative to DNA during transcription or DNA replication.
d. Hypersensitive sites exist -- these are the only sections of euchromatin that are not in nucleosomes.* See Becker fig. 23-18.
(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.
* We will assume that hypersensitive sites have no nucleosomes, unless the data in a particular experiment indicate otherwise.
To review the differences in states of chromatin, uses of DNase, etc., try the rest of 4-3, 4-9 & 4-10.
III. 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?
1. Need multiple steps not found in prokaryotes
a. Must de-condense (loosen up) euchromatin to a transcribable state = relatively loose (compared to heterochromatin and compared to inactive euchromatin). Pull out 30nm fiber to beads-on-a-string stage?
b. Many transcription factors (TF's) must bind to DNA first -- before RNA polymerase binds.
c. Polymerase must bind to TF's (not directly to the DNA) to get actual transcription.
2. What changes state of chromatin? (To tighten or loosen.)
a. Remodeling proteins: these are responsible for moving and/or loosening up nucleosomes. See Sadava 14.17 (14.16). These may be a separate set of proteins or the TF's that activate the modifying enzymes.
b. Enzymes that modify histone tails. Changes in modification may have a direct effect and/or affect binding of regulatory proteins. Some examples:
(1). 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. (Should kinases help chromatin get looser or tighter?)
(2). Acetylation of lys side chains of histones. Acetylation of histones → more active, looser chromatin. Acetylation of H3 & H4 is higher in active chromatin.
(3). Methylation -- Effects depend which amino acid side chains in which position of the protein are methylated -- some modifications increase likelihood of transcription, and some decrease it. (DNA can be methylated too; see last lecture.)
3. What triggers the tightening or loosening process? Do TF's come first or remodeling/modification enzymes? Current Model
a. Regulatory TF's (activators) bind first -- that triggers remodeling, modification, etc. Loosens up the chromatin in the area to be transcribed.
b. Basal TF's bind later -- After chromatin is loosened up, basal TF's (& possibly more regulatory TF's) can bind to the DNA, pol II can bind to the TF's, and transcription occurs.
4. How does this fit with the DNase sensitivity results?
a. Loosest -- Regions where transcription factors bind -- have nucleosomes removed &/or very loosened up = hypersensitive sites.
b. Looser -- Regions being transcribed -- have nucleosomes somehow "loosened up" or "remodeled" but not removed.
c. Loose -- Regions not being transcribed -- have regular nucleosomes ('loose', relative to heterochromatin, but 'tight' or 'not so loose' compared to transcribed euchromatin.) Regions that are not transcribed are often in euchromatin, not in heterochromatin.
IV. Details of transcription in eukaryotes (as vs. prokaryotes) See Becker Ch 21, pp 660-664 (665-670).
A. More of everything needed for transcription in eukaryotes.
1. Multiple RNA Polymerases (see last lecture). We will focus on pol II (makes mRNA).
2. More proteins -- Need TF's, not just RNA pol.
3. More Regulatory Sequences -- many dif. ones bind dif. TF's
4. An Overview & Some terminology
a. Control elements/sequences -- cis vs trans acting. (See 2nd table on handout 8A.)
Cis-acting regulatory element = affects only the nucleic acid molecule on which it occurs. Usually is a DNA sequence that binds some regulatory protein.
Trans-acting regulatory element = affects target nucleic sequences anywhere in the cell. The regulatory sequence codes for a regulatory molecule -- usually a protein -- that binds to a target -- usually a DNA sequence.
The term "trans acting" can be used to refer to the regulatory molecule (usually a protein) or to the DNA sequence that codes for it.
b. How Trans-acting and Cis-acting elements work together
Cis acting elements = DNA itself = same in all cells of multicellular organism = target of trans acting regulatory molecules.
Trans acting regulatory molecules = product of DNA = TF's & other molecules = different in different cell types and at different times.
In euk. the number of different types of cis and trans acting control elements is much larger than in prokaryotes. What are they like? See below.
c. Regulatory Proteins -- Positive vs Negative Control. See 1st table on handout 8A.
Regulation can be "+" or "-" depending on the function of the protein
Negative control -- If regulatory protein blocks transcription.
Positive control -- If regulatory protein enhances transcription.
Euk vs. Prok. -- Negative control (use of repressors) seems to be more common in prok.; positive control (use of activators) more common in euk. (See 1st table on handout 8A)
How you tell positive and negative control apart -- by effects of deletions.
B. Details of regulatory (cis acting) sites in the DNA. Prokaryotes have promoters and operators. What sequences do eukaryotes have in the DNA that affect transcription? (The following discussion refers mostly to regulation of transcription by RNA pol II. See texts esp. Becker for details about promoters etc. for pol I & III.) See Sadava Fig. 14.14 (14.13) or Becker fig.23-21 or handout 8A for structure of regulatory sites for a typical protein coding gene. Three types of regulatory sites:
1. Core Promoter
a. Numbering. Position of bases is usually counted along the sense strand from the start of transcription.
(1). "Start" = Point where transcription actually begins (usually marked with bent arrow) = zero.
(2). Upstream and Downstream
(a). Downstream = Going toward the 3' end on sense strand = in direction of transcription)
(b). Upstream = Going toward 5' end on sense strand = in opposite direction from transcription.
(3). Numbering -- some examples
(a). +10 = 10 bases downstream from start = 10 bases after start of transcription.
(b). -25 = 25 bases upstream from start = 25 bases before reaching start of transcription.
(c). +1 = first base in transcript; one that gets a cap (modified base attached to 5' end).
(4). Numbering -- misc. features
(a). There is no 'zero' base, just as there is no 'zero' year between BC and AD and no zero hour between am and pm.
(b). In some cases, the position of bases is counted along the sense strand from the start of translation. If it is done this way, the A in the first AUG is +1. However, numbering is assumed to be from the start of transcription unless specified otherwise.
(c). TF's, RNA pol, etc. bind to grooves in double stranded DNA, not to one strand. However, positions in the DNA are usually specified in terms of the sense strand only. This does NOT mean that the protein binds only to the sense strand.
b. Core Promoter Itself Core promoter is defined by what you need to allow RNA polymerase to start in the right spot. What is included in it?
(1). Actual point for start of transcription (where bent arrow is) plus a few bases on either side of 'start.' Usually includes a few bases of the 5' UTR (untranslated region).
(2). Binding sites: Part where basal TF's and RNA polymerase binding starts -- usually section just upstream (before) start point. Often includes short sequence called a TATA box (usually about 25 bases before start point).
(3). Additional Features: Often includes some additional or different sequences besides those specified. Not all promoters of Pol II are the same. (If you are interested in details, see Becker 21-12b (13 b), or 23-21)
2. Proximal Control Elements. (Proximal = Near).
a. Location: Near core promoter and start of transcription; usually "upstream" (on 5' side of start of transcription.) Usually includes regulator elements up to -100 or -200 (bases).
b. Terminology: Sometimes considered part of core promoter.
c. Function: Binding of appropriate proteins promotes or inhibits transcription. Identified by effects of deletions. Sequence and mechanism of action varies.
3. Distal Control Elements (Distal = Far)
a. Two kinds: Enhancers & silencers. These control elements can decrease (silencers) or increase transcription (enhancers).
b. These can be quite far from the gene they control (in either 5' or 3' direction = upstream or downstream). Can be in introns or in untranscribed regions.
c. These can work in both orientations -- Inverting them has no effect, unlike with promoters. See Becker fig. 23-22.
d. Mechanism of action -- bind TF's; see below.
4. Terminology & Misc. Details -- this is for reference; may not be discussed in class.
a. Boxes = short sequences that are found in regulatory regions (ex: TATA box)
b. Consensus sequences = sequence containing the most common base found at each position for all sequences of that type. Any individual version of sequence is likely to be different from the consensus at one or more positions. (Ex: TATAAAA = consensus sequence for TATA box. Means T is most common base in first position, A is most common in second position, etc.)
c. For multicellular organisms, term "operator" is not used for site/DNA sequence where a regulatory protein sits. Why? Because no polycistronic mRNA & no operons in higher eukaryotes. (Are some in unicellular euk.)
C. How do Basal Transcription Factors work?
1. Same in all cells. Needed to start transcription in all cells. See Sadava fig. 14.13 (14.12) or Becker fig. 21-13 (21-14).
a. Many basal TF's needed.
b. Basal TF's for RNA pol. II.
(1). Terminology: Basal TF's for pol II are called TFIIA, TFIIB, etc.
(2). Major one is TFIID; it itself has many subunits. Most studied subunit is TBP (TATA binding protein -- See Becker fig. 21-14 (21-15).) Recognizes TATA box when there is one.
(3). Other polymerases have TF's too, but TF's for pol II are of major interest, since pol II → mRNA
c. Basal TF's bind first to core promoter, and then RNA pol binds to them. Takes a lot of proteins to get started. RNA polymerase does not bind directly to the DNA.
D. How do Regulatory or Tissue Specific TF's Work?
1. Different ones are used in different cell types or at certain times. Not all are needed in all cells. See Becker fig. 23-24.
a. Bind to areas outside the core promoter -- usually to enhancers or silencers (distal control elements) but sometimes to proximal control elements
b. When regulatory TF's bind, can decrease or promote transcription.
(1). Activators. TF's called activators if bind to enhancers and/or increase transcription.
(2). Repressors. TF's called repressors if bind to silencers and/or decrease transcription.
c. How regulatory TF's affect transcription: DNA thought to loop around so silencer/enhancer is close to core promoter. TF's on enhancer help stabilize (or block) binding of basal TF's directly or indirectly to core promoter. (See Becker fig. 23-23 or Sadava 14.14 (14.13) and section on regulatory TF's below.)
d. Euk. vs Prok. repressors -- both 'repressors' interfere with transcription, but mechanism of action is different.
e. Role of Co-activators -- Proteins that bind to TF's on the enhancer and influence transcription (but don't bind directly to the DNA) are often called co-activator (or co-repressor) proteins. There are 2 ways co-activators affect transcription:
(1). Act as mediator -- Connect two parts of the transcription machine. One part of mediator binds to TF (which is bound to enhancer or silencer) and other part of mediator binds to basal transcription factors (or pol II) on core promoter and/or proximal control elements. Mediator = usual name of complex of co-activators that act this way.
(2). Modify state of chromatin. Bind to TF on enhancer and loosen up chromatin in gene to be transcribed. Remodeling proteins and histone modifying enzymes are included in this category.
To review gene structure & TF's, try problems 4R-2, 4R-5A & 4R6-A.
f. Co-ordinate control. A group of genes can all be turned on or off at once in response to the same signal (heat shock, hormone, etc.).
(1). Prokaryotes vs. Eukaryotes: Both prok. and euk. exhibit co-ordinate control, but mechanism is different. (See table below.)
(2). Location of coordinately controlled genes
(a). In prokaryotes, coordinately controlled genes are located together in operons.
(b). In eukaryotes, coordinately controlled genes do not need to be near each other -- they just have to have the same (cis acting) control elements. See Sadava 14.16 (14.14).
(3). Control elements:
(a). All genes turned on in the same cell type and/or under the same conditions share the same control elements -- therefore these genes all respond to the same regulatory TF's. Result is multiple mRNA's, all made in response to same signal (s).
(b). Most genes have multiple (cis acting) control elements. Therefore transcription of most genes is affected by more than one TF.
(c). Transcription of any particular gene depends on the combinations of TF's, not just one, available in that cell type.
(4). Differences in TF's. Different cell types make different regulatory TF's. Therefore different groups of coordinately controlled genes are turned on/off. See Becker fig. 23-24.
(5). Comparison of situation in prokaryotes vs multicellular eukaryotes:
Coordinately controlled genes are
Messenger RNA is
Polycistronic (1 mRNA/operon)
Moncistronic (1 mRNA/gene)
Control elements are found
Once per operon
Once per gene
|Control can be positive or negative but is more often||Negative -- repressors needed to turn gene off||Positive -- activators needed to turn gene on.|
Next time: Wrap up of Regulation of Eukaryotic Gene Expression. Then post transcriptional regulation -- what happens after transcription to control protein amount and/or activity?