BMF 2: L. 8 Genes, Chromosomes and DNA Packaging
What are condensins/cohesins?
*Condensins* and *Cohesins* belong to family of *SMC proteins* (structural maintenance of chromosome) They promotes condensation by creating physical contact between segments of DNA that may otherwise be distant on their/or on another chromosome. SMCs function in defined pairs: t*SMC1-SMC3* form *Cohesins*, while *SMC2-SMC4* form *Condensins*.
What are the orders of packaging of a chromatin fiber?
*First order packaging*: DNA wraps around histone proteins forming nucleosomes (euchromatin), called the *bead-on-a-string model*-10nm fiber. *Second order packaging*: Multiple histones wrap into a *30 nm fibre* consisting of nucleosome arrays in their most compact form (heterochromatin). Contains *linker histones* (H1). *Higher order packaging*: DNA packaging of the 30 nm fibre into the metaphase chromosome (utilizes condensins, cohesins and protein scaffold)
What are Heterochromatin and Euchromatin?
*Heterochromatin* are highly condensed chromatin, that are not transcriptionally active. 7-10% is always in this state. *Euchromatin* are less condensed chromatin, that are transcriptionally active
What are the properties of histones?
*Histones* are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into nucleosomes. Histones are rich in basic amino acids *Lys and Arg* giving them a positive charge to interact with DNA, which is negatively charged. Five major families of histones exist: H1/H5, H2A, H2B, H3, and H4. Histones H2A, H2B, H3 and H4 are known as the core histones and make up the octameric core of nucleosomes, while histones H1 and H5 are known as the linker histones
What are lampbrush chromosomes? What are their properties?
*Lampbrush chromosomes* are a special form of chromosome found in the growing oocytes of most animals, except mammals. Their features include: -Largest known chromosome -Actively transcribing DNA to RNA - Gene present in DNA loop are actively expressed -One loop can have 50 to 200Kbp - -loops extends away from chromosome to become active
What is the position effect?
*Position effect* is the effect on the expression of a gene when its location in a chromosome is changed, often by translocation. Genes moving towards heterochromatin gets inactivated (off), while going away from it gets activated (turned on)
How many base pairs of DNA are associated with each nucleosome?
200bp of DNA is associated with each nucleosome: wrapped DNA 147bp and linker DNA 53bp.
What are nucleosomes?
A *nucleosome* is a basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight (octameric) histone protein cores which consists of two units of H2A, H2B, H3, H4. DNA (about 147bp) is wrapped around this octamer core and makes contact with histone at minor grooves.
What is a bromodomain?
A bromodomain is an approximately 110 amino acid protein domain that recognizes acetylated lysine residues, such as those on the N-terminal tails of histones. Bromodomains, as the "readers" of lysine acetylation, are responsible in transducing the signal carried by acetylated lysine residues and translating it into various normal or abnormal phenotypes
What is a chromodomain?
A chromodomain is a protein structural domain of about 40-50 amino acid residues commonly found in proteins associated with the remodeling and manipulation of chromatin
What is a protein domain?
A protein domain is a conserved part of a given protein sequence and (tertiary) structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural domains.
What do chromatin-remodeling complexes do?
ATP-dependent chromatin-remodeling complexes regulate gene expression by either moving, ejecting or restructuring nucleosomes. These protein complexes have a common ATPase domain and energy from the hydrolysis of ATP allows these remodeling complexes to *reposition* (slide, twist or loop) nucleosomes along the DNA, *expel* histones away from DNA or facilitate *exchange* of *histone variants*, and thus creating nucleosome-free regions of DNA for gene activation
What is Chromatin? What are the functions of chromatin?
Chromatin is a complex of macromolecules found in cells, consisting of DNA, protein, and RNA. The primary functions of chromatin are: 1) to package DNA into a smaller volume to fit in the cell, 2) to reinforce the DNA macromolecule to allow mitosis, 3) to prevent DNA damage 4) to control gene expression and DNA replication. Chromatin is only found in eukaryotic cells. Prokaryotic cells have a different organization of their DNA (called genophore and is localized within the nucleoid region).
What is chromatin remodeling?
Chromatin remodeling is the dynamic modification of chromatin architecture to allow access of condensed genomic DNA to the regulatory transcription machinery proteins, and thereby control gene expression. Such remodeling is principally carried out by 1) *covalent histone modifications* by specific enzymes, i.e., histone acetyltransferases (HATs), deacetylases, methyltransferases, and kinases 2) *ATP-dependent chromatin remodeling complexes* which either move, eject or restructure nucleosomes with *variant histone subunits* that impart special properties to the chromatin
How does DNA carry genetic information? What are genes/genome?
DNA encodes information in sequences. These sequences are organized into discrete units called *genes* which are considered *heredity*. Genes contain 3-letter DNA code which connect to 3-letter RNA codes that correspond to 20 differnt amino acids, which build proteins. Complete DNA is called *genome* which consist of many genes and regulatory elements all of which are enclosed in the cell nucleus (not in prokaryotes).
How is DNA packaged in eukaryotes?
DNA is arranged in the cell nucleus with the help of *histones* to form the basic level of DNA compaction, called the *nucleosome*. A histone protein, called *Linker histone H1* binds the DNA between nucleosomes and facilitates packaging of the 10 nm "beads on the string" nucleosomal chain into a more condensed 30 nm fiber.
Why does DNA wraps around histone octamer core?
DNA wrapping around a histone octamer core is facilitated by runs of two or more A=T base pairs (which facilitate the bending of DNA) whereas runs of two or more GC base pairs have the opposite effect.
What is the state of chromosomes during interphase, prophase, and metaphase?
During Interphase, chromosomes are amorphous (without a clearly defined shape or form). In Prophase, chromosomes begins to condense, and by metaphase, they are maximally condensed. At this point, each DNA molecule is ~10,000-fold shorter than its extended length. After cell division, they become amorphous again.
What are the properties of chromatin during interphase? What are euchromatin/heterochromatin?
During interphase, the chromatin is structurally loose to allow access to RNA and DNA polymerases that transcribe and replicate the DNA. The local structure of chromatin during interphase depends on the genes present on the DNA.
What are chromosomal territories?
Each chromosome maintains its individuality and occupies a limited space in the nucleus called a *chromosome territory* (not true in bacteria, which lack a nucleus, or in some lower eukaryotes). When a gene needs to be turned, it must move to the a transcriptionally active neighborhood.
How do chemical modifications of structural proteins affect chromatin structure?
Epigenetic chemical modification of the structural proteins in chromatin alters the local chromatin structure, in particular chemical modifications of *histone proteins* by *methylation* and *acetylation*. Heterochromatin (condensed/inactive) are associated with methylation and have been silenced through histone deacetylation.
How are genes organized on a chromosome?
Genes are organized on a chromosome by a nucleotide sequence which depicts gene arrangement and sequence linearity. Humans have 3.2x109 nucleotides which make up 23 different chromosomes. Chromosomes are made up of a complex of DNA and proteins called *chromatin*. Each person has two copies of each chromosome (homologous chromosome) one from each parent. The only nonhomologous pair is male sex chromosome X and Y.
What is Genomic DNA? What are its properties?
Genomic DNA refers to the DNA constituting the genome of a cell or organism. Genomic DNA is organized into linear or circular *chromosomes*. It is linear in the nucleus of an eukaryotic cell and circular in the mitochondria or chloroplast of an eukaryotic cell or in prokaryotes.
How are 30nM fibers condensed to make chromatid?
Higher orders of DNA packaging are not well understood. The 30 nM fiber is looped onto a protein scaffold. These looped regions are variable in size. Beginning in *prophase*, the loops and scaffold are helically packed to form *chromatid*. Proteins called *Condensins* are necessary for this level of packaging.
How is histone H1 related to 30nm fibers?
Histone H1 is an integral part of 30nm fiber. H1 has two DNA binding sites, one binds the linker DNA and the other binds the DNA wrapped around the histone octamer. A nucleosome and a linker Histone H1 make up a *chromatosome*
What do histone acetyltransferases (HATs) do?
Histone acetytransferases acetylate specific lysine residues, increasing accessibility to DNA, which is necessary for transcriptional activation.
What is the Histone Code?
Histone modifications serve to recruit other proteins by specific recognition of the modified histone via protein domains (*histone code readers*) specialized for such purposes, rather than through simply stabilizing or destabilizing the interaction between histone and the underlying DNA. These recruited proteins then act to alter chromatin structure actively or to promote transcription through ATP-dependent chromatin remodeling
What do Histone Variants do? What are some examples of histone variants?
Histone variants alter DNA binding affinity. For example, *H3.3*, a variant of Histone H3, is assembled into octamers associated with genes that are actively transcribed. The presence of H3.3 might help stabilize chromatin in an open, transcriptionally active form. (Note: There are only 4 aa differences between H3 and H3.3 *CENPA* is a Histone H3-like variant which exclusively replaces conventional H3 in the nucleosome core of centromeric chromatin at the inner plate of the kinetochore. Required for recruitment and assembly of kinetochore proteins, mitotic progression and chromosome segregation: Maintaining kinetochore attachment. *H2AX*: A variant of H2A used in attracting DNA repair enzymes.
How are the structures of chromosome regulated?
Nucleosomes control the accessibility of the machinery associated with replication or transcription, called *Chromatin Remodeling*.
What are polytene chromosomes? What are their properties?
Polytene chromosomes are giant chromosomes common to many dipteran (two-winged) flies. Their features include: -DNA synthesis without cell division, which leads to polyploidy -Copies are held side by side, which leads to banding Dark bands (95%) (condensed) and light interbands (5%) (where transcription occurs). These chromosomes are an easy target to see histone modifications
What is required for nucleosomes to condense into 30nM fibers?
The *N-terminal tails* of core histones are required for nucleosomes to condense into the 30 nM fiber. These tails make contacts with adjacent nucleosomes stabilizing the fiber. There are two proposed models: *Solenoid* and *zigzag* both of which can be found at any given time.
How are N-terminal tails of histones associated with gene expression?
The N-terminal Tails of histones can be covalently modified to allow access of condensed genomic DNA to the regulatory transcription machinery proteins, and thereby control gene expression Certain enzymes covalently modify specific amino acid residues in the N-terminal tails of histones. These modifications play a specific role. An example includes *Histone acetyltransferases* (HATs). Covalent modifications are considered to be part of the *epigenome*, which are inherited characteristics that do not involve the nucleotide sequence of DNA. Modification includes, *phosphorylation*, *acetylation*, *methylation*, and *ubiquitination*
How does chromosome packaging changes in regions where genes are expressed?
The degree of packaging is less in chromosomal regions where genes are expressed. Less packaging allows *transcription factors* and *RNA polymerase* to gain access to the gene, allowing transcription to occur. Tightly packaged DNA prevents transcription and therefor gene expression.
What are the classes of chromatin remodeling complexes?
The three main classes of chromatin remodeling complexes are: 1. *SWI/SNF*: *bromodomain* associates with histones, and they *activate* transcription 2. *ISWI*: does not have a domain that associates with histones and *represses* transcription. (with the exception of *CHRAC*) 3. Mi2/NURD: *chromodomain* associates with histones and they *repress* transcription Bromodomain protein -> Stabilized open state Chromodomain protein -> Stabilized closed state
What do condensins/cohesins do?
These two proteins change the nuclear material from amorphous to a condensed state. *Cohesins* hold sister chromatids together from replication in the *S-phase* until anaphase when removal of cohesin (by *separase*) leads to separation of sister chromatids. *Condensin* is present within the cell nucleus during interphase and is involved in an early stage of chromosome condensation within the *prophase* nucleus and contributes to the assembly of condensed chromosomes, in which two *sister chromatids* are fully resolved.
What is the histone code/reader important? Give an example.
When covalently modified genes are replicated, half the new sequence is no longer modified. Histone code/code readers are used in order to maintain an open or closed chromatin state. To remain open *bromodomain HAT* is recruited, which results in the aceylation of the new strand To remain closed, *Heterochromatin Protein 1* (HP1) is recruited to maintain a closed state until methylation by *methyltransferase* can occur.