Part IV => DNA and RNA. 4.7 Gene Regulation 4.7a Chromatin Remodeling 4.7b Transcriptional Control

Transcription

1 Part IV => DNA and RNA 4.7 Gene Regulation 4.7a Chromatin Remodeling 4.7b Transcriptional Control

2 Overview of Gene regulation - The expression of the so-called housekeeping genes (eg basal transcription factors) occurs in a constitutive manner ie they are always on and expressed at a basal level so as to maintain basic cellular functions such as replication and repair of DNA - In contrast, the expression of many tissue-specific genes is tightly regulated such regulated genes (eg hormones) are needed only occasionally and they are turned on or off as the circumstances demand - Simply put, gene regulation is the process by which the cell controls: (a) When to turn on or off a gene (b) The rate at which to turn on or off a gene (c) The duration for which the gene is to be turned on - Such gene regulation is hierarchical in that it occurs at multiple levels ie it can be tweaked at various stages such as chromatin level or transcriptional level - In particular, chromatin remodeling and transcriptional control represent two key stages at which the expression of a gene can be tightly regulated

3 Hierarchy of Gene Regulation Chromatin Chromatin Remodeling DNA Transcriptional Control RNA 5 -Cap Poly(A) Intron Exon mrna 5 -Cap RNA Splicing Poly(A) mrna Degradation 5 -Cap Poly(A) Translation Post-translational Modification (PTM) Protein Degradation Protein

4 Section 4.7a: Chromatin Remodeling

5 Synopsis 4.7a - In eukaryotes, DNA does not exist in isolation but rather it is wound/wrapped (or negatively supercoiled) around bead-like protein complexes called histones to form what has come to be known as a nucleosome - Nucleosomes represent the first building blocks for packaging/folding DNA into a higher-order architecture called the chromatin - Chromatin does not only serve to package DNA into a highly condensed form but it also serves to protect the DNA as well as act as a gatekeeper to regulate the access of other proteins such as RNA polymerases and transcription factors, thereby tightly controlling the expression of genes - In order to allow gene expression in a regulated manner, chromatin must undergo transient opening and closing according to cellular demands such ability of chromatin to undergo dynamic and flexible structural changes is referred to as chromatin remodeling - Chromatin remodeling is under the control of a wide array of so-called chromatin-remodeling protein complexes that in part execute their function via covalent modification of histones and/or DNA

6 DNA Packaging: The Conundrum 40,000km How long is human DNA?! Is there really a need to package it into a condensed form? Within average human cell: Human genome size = 3.2 x 10 9 bp (from 46 DNA molecules/cell = 46 chromosomes/cell) Helical rise (assuming B-DNA) = 3.4Å/bp => 0.34nm/bp Contour (fully extended) length = (0.34nm/bp)*(3.2 x 10 9 bp) => 1.1 x 10 9 nm => 1.1m (1.1 x 10 6 m) Diameter of human cell nucleus = 10 m => 1.1m DNA thus has to be tightly packaged! Within average human body: Number of cells in human body = 10 x (~10 trillion) cell (cf: US$20x10 12 national debt!) Total length of human DNA = (10 x cell)*(1.1m/cell) => 11 x m => 11,000 x 10 6 km Celestial distances: Earth circumference Earth Moon Earth Sun 1nm = 10 Å 1m = 10 9 nm 1km = 5/8 mile = 0.04 x 10 6 km => Human DNA wraps around the earth ~300,000 times! = 0.40 x 10 6 km => Human DNA stretches back-and-forth ~30,000 times! = 150 x 10 6 km => Human DNA stretches back-and-forth ~100 times!

7 DNA Packaging: The Big Picture Heterochromatin highly-condensed and transcriptionally-inactive form of chromatin Euchromatin loosely-packed and transcriptionally-active form of chromatin

8 DNA Packaging: The Histones Histone Length / aa Mass / kd Arg / % Lys / % Basic / % H H2A H2B H H Histones are small proteins that engage in non-specific interactions with DNA (cf transcription factors), thereby allowing it to be packaged into a condensed form called chromatin with five major classes of histones being H1, H2A, H2B, H3, and H4 - How do histones interact with DNA? Histones are highly basic due to their evolutionary enrichment with basic amino acid residues such as Lys and Arg - The basic/alkaline character of histones is a perfect compliment to the negatively charged DNA a marriage surely made in heavens! - Histones are among the evolutionarily most conserved proteins across species eg histone H4 from cow and pea (species that apparently diverged over a billion years ago!) share a remarkable 98% identity at amino acid level - While H4 is the most conserved of all histones, H1 displays highest variability between species

9 DNA Packaging: The Nucleosome DNA (2nd turn) 90 2x H3 2 x H2A 2 x H4 Nucleosome Cartoon (H2A) 2 (H2B) 2 (H3) 2 (H4) 2 x H2B 2 DNA (1st turn) - Nucleosome represents the basic building block or unit of DNA packaging with a contour (extended) length of 80Å - A single nucleosome is comprised of a histone octamer made up of two copies of each of H2A, H2B, H3, and H4 around which double-helical DNA wraps around twice or makes two left-handed superhelical (coiled double-helix) turns involving ~200bp - Such negative supercoiling represents a compression of the contour length of B-DNA by nearly an order of magnitude 200bp equates to 680Å (200bp 3.4Å/bp) => 680Å/80Å=9 - H1 histone is not an integral component of nucleosome H1 docks at the base of the nucleosome close to the DNA entry and exit so as to serve as a glue or linker to bring individual nucleosomes together into a condensed form (eg the 30-nm fiber vide infra!) Nucleosome Structure Left-handed Turn (-ve) Right-handed Turn (+ve)

10 DNA Packaging: From DNA to Chromosome - The continuous left-handed winding of DNA onto multiple histone octamers yields a loosely defined 10-nm fiber with a beads-on-a-string appearance H1 histone not needed! 10 nm - The 10-nm fiber undergoes further condensation in the presence of H1 histone to form what is called the 30-nm fiber - Within the 30-nm fiber, the 10-nm fiber winds up into a solenoid-like coiled conformation (though it could also fold into the 30-nm fiber in a zigzag fashion) - Addition of non-histone scaffold proteins to the 30-nm fiber allows further compaction of DNA into a higher-order structural organization called chromatin - Chromatin (harboring 1.1m/46 24,000 m of double-helical DNA) finally packs into an X- shaped chromosome (transiently visible only during mitosis) roughly measuring 5 m-by-1 m

11 Chromatin Modification: Overview Temporal DNA only temporarily or transiently exposed at a time Spatial only specific sequences/regions within DNA exposed - In order to allow cellular processes such as RNA transcription, DNA replication, and DNA repair in a regulated manner, chromatin must undergo transient opening and closing such ability of chromatin to undergo dynamic and flexible structural changes is called chromatin remodeling - Chromatin remodeling is under the control of a wide array of so-called chromatin-remodeling protein complexes (eg SWI/SNF and RSC ) interactions between histones and DNA within nucleosomes are disrupted to render the DNA more accessible in a highly temporal and spatial manner - Simply put, chromatin remodeling complexes facilitate the transient release of DNA from histone octamers so that it can be accessed by other proteins and enzymes such as RNA polymerase to initiate gene expression how?!

12 Chromatin Modification: Epigenetic Control - DNA and histones within the chromatin are subject to a wide variety of chemical modifications that allow the chromatin to undergo remodeling and thereby directly impinge upon the ability of genes to be switched on or off - The study of such chromatin modifications executed by specific protein domains that form an integral part of chromatin remodeling protein complexes that affect the ability of genes to be expressed without altering the DNA sequence itself has come to be known as epigenetics epi meaning beyond or above - Two major epigenetic mechanisms involved in the control of gene expression are: (1) Histone modification (2) DNA methylation

13 Chromatin Modification: Histone Modification - Histone tails are highly decorated with residues such as Lys, Arg, Ser, Thr and Tyr - Many of these residues in histones are subject to post-translational modification (PTM) by enzymes that couple the extra/ intra-cellular needs of the cell with the state of its chromatin such that gene expression can be turned on or off in an epigenetic manner - Major histone PTMs include (in the order of prevalence): - Acetylation (Ac) of Lys residues - Methylation (Me) of Lys/Arg residues - Phosphorylation (P) of Ser/Thr/Tyr residues - Such PTMs not only alter the net charge on histones but also provide docking sites for the recruitment of chromatin remodeling complexes - Histone modification thus plays a central role in the alteration of histone-dna interactions reducing the positive charge on histones generally correlates with loosening of chromatin structure and thus facilitating gene expression and vice versa

14 Chromatin Modification: Histone Acetylation HATs HDACs Lysine (K) Acetllysine (AcK) - Acetylation on Lys is catalyzed by histone acetyltransferases (HATs) using acetyl-coa as an acetyl donor - Lys residues can be deacetylated by histone deacetylases (HDACs) - Histone acetylation reduces net positive charge on histones thereby mitigating histone-dna interactions, loosening up chromatin structure (usually favors euchromatin), and making it more accessible to RNA polymerase and transcription factors - Acetyl moiety on Lys residues in histone also serves as a docking site for bromodomain a highly ubiquitous protein domain found in transcriptional co-activators such as PCAF (a component of chromatin remodeling protein complexes) - Although histone acetylation generally correlates with transcriptional activation, it can also result in transcriptional repression depending on the context of target lysine

15 Chromatin Modification: Histone Methylation HMTs HMTs HMTs HDMs HDMs HDMs Lysine (K) Monomethyllysine (MeK) Dimethyllysine (Me 2 K) Trimethyllysine (Me 3 K) - Mono-, di-, or tri-methylation on Lys/Arg is catalyzed by histone methyltransferases (HMTs) using S-adenosylmethionine (SAM) as a methyl donor - Lys/Arg residues can be demethylated by histone demethylases (HDMs) - Unlike acetylation, histone methylation does not reduce net positive charge on histones thus it may loosen up chromatin (favor euchromatin) and make it transcriptionally-accessible, or alternatively, it may induce condensation of chromatin (favor heterochromatin) and render it transcriptionally-inaccessible - Methyl moieties on Lys/Arg residues in histone also provide docking sites for chromodomain a highly ubiquitous protein domain found in transcriptional co-activators and co-repressors such as HP1 (a component of chromatin remodeling protein complexes) - Histone methylation can correlate both with transcriptional activation and transcriptional repression depending on the location of Lys/Arg target residue and the state of modification of other residues within its vicinity

16 Chromatin Modification: Histone Phosphorylation Kinases Phosphatases Serine (S) Phosphoserine (ps) - Phosphorylation on Ser/Thr/Tyr residues is catalyzed by a plethora of protein kinases using ATP as a phosphoryl donor - Ser/Thr/Tyr residues can be dephosphorylated by protein phosphatases - Histone phosphorylation imparts negative charge on histones this can have profound effects on histone conformation leading to both chromatin condensation and relaxation - Phosphorylated tyrosine (py) residues in histone may also serve as a docking site for SH2 domain a modular component of many cellular proteins as well as transcriptional regulators - Histone phosphorylation can correlate both with transcriptional activation and transcriptional repression depending on the cellular context

17 Chromatin Modification: DNA Methylation Cytosine (C) 5-methylcytosine (M5C) - Of the four DNA bases, two are subject to methylation cytosine and adenine (adenine methylation only occurs in prokaryotes such as during DNA replication) - In eukaryotes, only cytosine base in DNA is subject to methylation particularly within CG-rich sequences called the CpG islands - Cytosine methylation is catalyzed by DNA methyltransferase (DNMT) using S-adenosylmethionine (SAM) as a methyl donor and releasing S-adenosylhomocysteine (SAH) as a by-product - Cytosine can also be demethylated by DNA demethylases SAM - Unlike the more subtle nature of histone modification, DNA methylation switches off eukaryotic gene expression by virtue of its ability to induce chromatin condensation

18 Chromatin Modification: Transcriptional Initiation Complex Transcription Initiation site Binding of TAF1 via its bromodomain to acetylated histones can result in the recruitment of basal transcriptional factors such as TBP required for the formation of transcriptional initiation complex at gene promoters

19 Exercise 4.7a - Explain why histones from different species are so similar - What is the role of histones in compacting DNA? - Compare the binding of histones and transcription factors to DNA - Describe the levels of DNA packaging in eukaryotic cells - Why is chromatin remodeling necessary for efficient gene expression? - List three major ways that histones can be covalently modified - Describe how histone modifications can affect the structure of nucleosomes and the function of transcription factors - Summarize the role of DNA methylation in epigenetic control

20 Section 4.7b: Transcriptional Control

21 Synopsis 4.7b - Since the transcriptional machinery is largely dependent upon transcription factors, modulating the activity of transcription factors presents a key step in the decision to turn on or off a target gene at transcriptional level - In particular, the so-called gene-specific transcription factors (as opposed to basal transcription factors) are subject to a wide array of post-translational modifications (PTMs) - Such PTMs not only upregulate but can also downregulate the activity of specific transcription factors depending on the state of the cell - PTMs thus tightly control protein-dna interactions pertinent to gene transcription - In particular, the modular design of transcription factors befits their role as regulators of gene transcription in that they not only become subject to PTM but may also recruit co-activators or co-repressors depending on the cellular needs

22 Modular Proteins - With the exception of small proteins designed for simple tasks, a vast array of more complex and regulatory proteins are not monolithic but rather modular ie they can be divided into constituent parts or regions specialized for specific roles - Such specialized parts/regions of modular proteins are referred to as modules, or more commonly as domains - Modular proteins are a hallmark of the eukaryotic cell by virtue of such domains, a single protein may not only accomplish multiple tasks but its function may also be tightly regulated eg ligand binding or PTM of one domain within a transcription factor may control the ability of its DNA-binding (DB) domain to affect the expression of a target gene

23 Transcription Factors: Modular Design Transcription Factor mrna DNA Promoter Gene - The DNA-binding (DB) proteins or Transcription factors harbor a set of well-defined structural motifs (as a constituent component of the so-called DB domains) such as: (1) Helix-Turn-Helix (2) Helix-Loop-Helix (3) Zinc Finger (4) Leucine Zipper - Such structural motifs usually bind to the major groove of DNA by virtue of their ability to recognize sequence-specific motifs (or cis-acting elements) located within target gene promoters - Because of the (pseudo)palindromic nature of cis-acting elements within gene promoters, DB domains usually bind to DNA as dimers one monomer recognizes the element on one strand and the other on the opposite within the major groove of the double helix! - Such dimeric interaction also accounts for the high specificity of protein-dna interactions in that the binding of two monomers simultaneously (and usually symmetrically) doubles the free energy - Upon binding to DNA promoters via their DB domains, transcription factors activate or repress expression of target genes, usually by recruiting and interacting with other cellular proteins termed co-activators and co-repressors

24 Transcription Factors: Ligand-Activated - SHRs usually exist as monomers in complex with heat shock proteins (HSPs) in the cytoplasm Steroid Hormone - After diffusion through the cell membrane, the binding of the hormone to the LB domain results in its dimerization LB DB + LB DB LB LB SHR DB DB - Dimeric SHR translocates to the nucleus and binds to the target gene promoters via its DB domain TA HSPs TA Nucleus TA TA - Recruitment of cellular factors required to assemble the transcriptional machinery at the gene promoters is aided by the LB and TA domains - This turns on gene expression of specific proteins which in turn set about causing changes to the cell in response to the hormone DNA TA LB LB DB DB TA Transcriptional machinery mrna

25 Transcription Factors: PTM-Activated - Ligand binding to RTK induces receptor dimerization and/or autophosphorylation Ligand - Activated RTK serves as a binding site for the recruitment of adaptors such as GRB2 (via its P SH2 domain) to the inner membrane surface P (IMS) in a phosphorylation(tyr)-dependent manner - Since GRB2 adaptor exists in complex with SOS exchange factor, the recruitment of guanine nucleotide exchange factor SOS to the IMS catalyzes GDP-GTP exchange in Ras GTPase, thereby resulting in its activation - Next, activated Ras binds and activates Raf kinase - Raf kinase then activates the kinase MEK via Ser/Thr phosphorylation RTK P P Extracellular SH2 GRB2 Jun SH3 SH3 SOS Ras GDP Cytoplasm Nucleus MAPK Ras GTP MAPK P MEK P Raf P mrna - This is followed by the activation of the MAP kinases (MAPKs) such as ERK2 by MEK, also via Ser/Thr phosphorylation P Jun - Activated MAPK translocates to the nucleus and phosphorylates specific transcription factors (eg Jun/Fos/Myc) - Phosphorylated Jun binds to its promoter within the target genes and turns on gene expression of specific proteins which in turn set about causing changes to the cell in response to the ligand

26 Transcription Factors: (1) Helix-Turn-Helix turn Helix-Turn-Helix (HTH) CAP RESPONSE ELEMENT 5 -AATGTGATCTAGATCACATT-3 3 -TTACACTAGATCTAGTGTAA-5 - HTH motif is usually comprised of two consecutive -helices interrupted by a turn - Within the inter-helical turn, the polypeptide chain adopts an extended and ordered structure so as to reverse its direction and allow the two helices to pack together - Examples include bacterial CAP (catabolite activator protein) and viral (Lambda repressor) Two HTH motifs (blue and red helices) binding to two consecutive major grooves

27 Transcription Factors: (2) Helix-Loop-Helix HIF1 RESPONSE ELEMENT 5 -ACGTG-3 3 -TGCAC-5 Helix-Loop-Helix (HLH) Two HLH motifs (green and cyan helices) binding to a major groove - HLH motif is usually comprised of two consecutive -helices interrupted by a loop - Within the inter-helical loop, the polypeptide chain harbors a highly flexible or disordered structure so as to allow the two helices to fold back onto each other (without the polypeptide chain necessarily having to reverse its direction!) - Examples include HIF1 (hypoxia-inducible factor 1) and Myc (myelocytomatosis oncogene homolog)

28 Transcription Factors: (3) Zinc Finger ER RESPONSE ELEMENT 5 -AGGTCANNNTGACCT-3 3 -TCCAGTNNNACTGGA-5 Zinc Finger (ZF) Two ZF motifs (green and blue) binding to two consecutive major grooves - ZF motif is usually comprised of a Zn 2+ divalent ion sandwiched between a two-stranded antiparallel -sheet ( -hairpin) and an -helix ( topology) - Zn 2+ ion is tetrahedrally coordinated by four ligands in the form of either four cysteine residues (C4-type) or two cysteine and two histidine residues (C2H2-type) - Examples include nuclear/steroid receptors such as ER (estrogen receptor ) and EGR1 (early growth response 1)

29 Transcription Factors: (4) Leucine Zipper - LZ motif is usually characterized by a leucine residue at every seventh position within 4-5 successive heptads of amino acids with each of two constituent polypeptide chains - Inter-chain van der Waals contacts and ionic interactions drive dimerization of the two amphipathic LZ polypeptide chains so as to enable them to adopt -helices packed against each other - LZ motifs harbor a highly basic region (BR) at the N-terminus that enables them to adopt continuous -helices and wrap around each other into a coiled coil - LZ motifs together with their BR segments are collectively referred to as basic zipper (bzip) domains - While bzip domains contact the major groove within the DNA via their BR segments, LZ motifs are critical for their structural integrity - Examples include Jun and Fos collectively referred to as activator protein 1 (AP1) LZ BR LZ BR AP1 RESPONSE ELEMENT 5 -TGACGTCA-3 3 -ACTGCAGT-5 Leucine Zipper (LZ) Two bzip domains (yellow and green) binding to a major groove

30 Transcription Factors: Electrostatic Polarization ZFI Positive charge ZFI ZFI Negative charge Neutral charge ZFII 90 ZFII ZRE duplex 90 ZFII ZFIII ZRE duplex ZFIII ZFIII ZRE duplex Front View Side View Back View Electrostatic surface potential map of a DB domain in complex with DNA - DB domains are basic proteins in that they carry a net positive charge with pk values typically greater than 9 and so they should be if they are to be attracted to a negativelycharged DNA! - Not only are they basic but DB domains generally tend to be electrostatically polarized they harbor a net positive charge on one face of the molecule that docks into the major groove (the minor groove is too narrow and thus spatially-restrictive) of DNA - Ionic (not electrostatic!) interactions thus play a key role in driving protein-dna interactions though van der Waals contacts and hydrogen bonding by and large account for the specificity of such unions of the opposites

31 Exercise 4.7b - Describe the types of interactions between nucleic acids and proteins - Compare the major structural motifs within transcription factors involved in the recognition of DNA - Why do transcription factors usually bind to DNA as dimers? Why do they only bind to the major groove?