Module 3 Sections 6-8 Flashcards
genome structure and formatting, chromosome organization and packaging, chromatin remodeling and histone modifications
genomes
complete set of genetic material encoded in a cell or virus
what do genomes contain
1 set of autosomes and 2 sex chromosomes
contains both coding and non-coding information, both functional and non-functional components of DNA
prokaryotic genomes
mostly functional DNA
eukaryotic genomes
mostly non-functional DNA
functional DNA in the genome
highly conserved because it improves an organism’s fitness
non-functional DNA in the genome
has no known biological contributions
coding DNA (from functional DNA)
codes for a specific protein
non coding DNA (from functional DNA)
does not code for a protein
the human genome has:
3 billion nucleotide base pairs
23 pairs of chromosomes (22 autosomes and 2 sex chromosomes (1 pair))
estimated 20,000-25,000 genes
genomic complexity
analysis and comparison of different genomes of the 3 major domains of life
3 major domains of life
- bacteria
- archaea
- eukaryotes
bacteria domain
Small prokaryotic microorganisms
Have a plasma membrane but no internal organelles or nucleus
Have a genome consisting of a single, circular DNA molecule that is several million base pairs long
First genome sequenced was the bacterium Haemophilus influenzae
archaea domain
Like bacteria, unicellular organisms with no internal organelles or nucleus
Similar appearance to bacteria, but are more closely related to eukaryotes with respect to some genes and metabolic pathways
Have many species that thrive in extreme environments of high ionic strength, high temperature, or low pH
eukaryote domain
Unicellular or multicellular organisms with cells having a membrane-bounded nucleus, multiple chromosomes and internal organelles
Genomes larger than bacteria and archaea with billions of nucleotides
Orthologs (genes of different species that evolved from a common ancestor)
the human chromosome
arranged into 46 chromosomes
ranging from 50-250 million base pairs of DNA
22 homologous pairs of autosomes and 2 sex chromosomes or 1 pair (females have 2 X, males have 1 X and 1 Y)
human chromosome structure
P-arm (shorter)
Q-arm (longer)
Centromere
human chromosome banding pattern
Unique band pattern when stained with a dye called Giemsa
Dark bands are heterochromatin, areas which stain heavily. It is the condensed portion of chromosomes that are not transcriptionally active
Lighter bands are euchromatin, which stains poorly or not at all. These regions are the genes that are being actively expressed
humans vs primates similarities
shared a common ancestor around 7 million years ago
humans vs primates differences
apes = 24 pairs of chromosomes
humans = 23 pairs
humans have one chromosome 2 (result of end-to-end fusion of 2 ape chromosomes)
primates have two chromosome 2s
genomic differences in 2 types:
1. single nucleotide polymorphisms SNPs
2. large genomic rearrangements
single nucleotide polymorphisms (SNPs)
Genomic base pair change that helps distinguish one species from another
Most common type of genetic variance among different people
Many or may not result in an amino acid change
large genomic rearrangements
Larger alterations within the DNA sequence of chromosomes
Inversions:
- A mutation that results from the inversion of a large segment of DNA in a chromosome
- May be as a result of a segmental duplication, transposition of one copy to another arm of the same chromosome and recombination between the 2 segments
Fusions
- The rearrangement of chromosomal DNA by deletion, duplication, insertion, or transposition to form a hybrid gene
outgroups
a way to compare genome sequences with those of more distantly related organisms
comparative genomics
researchers assign gene functions by comparing the genomic features of different organisms - can be done with DNA, RNA or protein
homologs
2 genes with a demonstrable sequence similarity, whether or not they are closely related by function
Implies an evolutionary relationship
Sequence similarity and a functional relationship go hand-in-hand
orthologs
Possess a clear sequence and functional relationship to each other
Genes derived from an ancestral gene in the last common ancestor of these 2 species
paralogs
Genes that are similarly related to each other but within a single species
Arise most often from gene duplication in a single genome, followed by specialization of one or both copies of the gene over the course of evolution
the gene unit
a single gene is composed of a promoter sequence which defines where transcription will begin, exons and introns
splicing
process of removing introns from a primary RNA transcript
why do simpler organisms not use RNA splicing?
Alternative splicing allows multiple, functionally distinct proteins to be encoded by a single gene. This increases protein diversity. Splicing can be specific too certain tissues, conditions, and developmental states. Bacteria and simpler organisms lack intros, and do not undertake alternative splicing. This causes bacteria to lack a level of diversity found in eukaryotes
chromosome packaging must:
- be highly organized
- allow access to factors that regulate DNA replication
- allow access to factors that regulate transcription
levels of organization (smallest to biggest)
nucleotides, DNA double helix, histones, nucleosomes, chromatin, mitotic chromosome
histones
largest protein component of chromatin
basic, positively charged proteins that assemble into octamers - each octamer unit contains 2 copies of the 4 different histone subunits
DNA and histones confirmation
DNA is wrapped twice around the histone octamers - the positive charge of the histone protein allows them to interact with the negatively charged DNA backbone throuhg electrostatic interations and forms a structure called a NUCLEOSOME
organization of the core histones
H3 and H4 form a heterotetrameter
each histone octamer has 2 copies of each histone - H2A, H2B, H3, H4
H3 and H4, H2A and H2B is a tetramer that assemble together into an octamer
H1 unit
The DNA that is not wrapped around the histone octamer serves as a linker between nucleosomes. This linker binds the histone H1 unit
H1 binds to the nucleosome and protects the linker DNA from degradation
what are histones made up of
rich in arginine and lysine, making up 25% of any histone protein
nucleosome
When the histone octamer binds DNA, it forms a left-handed solenoidal supercoil (an over-wound DNA strand, forming a tightly packed helical structure)
nucleosome structure
Nucleosome structure provides a 6-7 fold compaction of DNA
The DNA is not uniformly bent, but instead follows a pattern of relatively straight 10 base pair segments joined by bends
the histone-fold motif
A motif for folding is composed of a globular domain that consists of a 3 alpha-helices linked by 2 short loops
structural unit of a nucleosome
Basic structural unit is composed of a head-to-tail dimer of histone-fold motifs
Due to their 3D structure, each histone-fold dimer forms a V-shaped structure that has 3 DNA-binding sites
The nucleosome will interact with DNA via the minor groove, at all 3 DNA-binding sites
AT base pairs influence on nucleosome binding
Presence of consecutive A-T (2) and G-C (3) base pairs can influence the ability of DNA to bind the nucleosome
A local abundance of A-T base pairs in the minor groove (where it is in contact with the histones). Facilitates the compression of the minor groove that is needed for tight wrapping of DNA around the histone octamer
ping of DNA around the histone octamer
Histone octamers assemble well with sequences where there is 2 or more A-T base pairs staggered at 10 bp intervals, because DNA is naturally bent at these sequences. A-T base pairs are spaced along the same face of the helix, and DNA bends into a circle
GC base pairs influence on nucleosome binding
Tracts of G-C base pairs have opposite effects to A-T, where they prevent compression of the minor groove, and are preferred at positions not facing nucleosomes
The intermediate
DNA –> Nucleosome –> 30 nm filament
DNA in the intermediate
tightly wraps around an octamer of the histone proteins
nucleosome in the intermediate
the complex of DNA and a histone octamer
H1 binds to the nucleosome in the DNA linker region
30 nm filament in the intermediate
after binding of H1, nucleosomes condense into a compact filament with a width of 30 nm
thought to exist in living cells but hasn’t been visualized or proven
how does H1 bind to the nucleosome?
H1 has 2 DNA-binding sites - 1 to the arm of linker DNA and the other to the central region of the DNA strand in the nucleosome
only one H1 subunit is present per histone octamer unlike the other core histones
chromatosome
when H1 is bound to the histone octamer and DNA
progressive levels of DNA organization in coils
DNA –> nucleosome –> 30 nm filament –> extended form of chromosome –> condensed section of chromosome –> mitotic chromosome
evidence that DNA is packaged into regularly organized repeating units
- Kornberg treated chromosomal DNA with a nonspecific DNA nuclease, called micrococcal nuclease, that cuts DNA wherever it is not associated with proteins. The fragments produced were separated by size on an agarose gel
- If DNA is packaged by proteins into units of a particular size, the nuclease would cleave only the DNA between these units, and the protected DNA segments (bunded to protein) would migrate in the gel as a ladder of unit-sized bands
- If proteins were distributed on DNA in a random way, then nuclease digestion would produce a smear of DNA fragments with no regular pattern - Protected DNA segments migrated on the gel as a ladder of regularly-spaced bands
- Suggests that DNA is packaged by proteins into units that encompass approx. 200 base pairs
things to consider for DNA compaction
- dynamic
- modifiable
- responsive
dynamic in DNA compaction
DNA compaction must be dynamic, meaning changes in the degree of condensation must occur quickly and when needed, as cell passes through the stages of the cell cycle
When in its highly compacted form, DNA is not accessible to transcription or replication enzymes so it must be able to rapidly expose regions containing genes that are required at any given moment, and then condense again
modifiable in DNA compaction
DNA compaction must be globally and locally modifiable
Global = modifications for processes like mitosis or replication
Local = giving access to specific genes for transcriptional regulations
responsive in DNA compaction
Must be able to respond to modification enzymes that are able to alter the state of DNA condensation
Enzymes can target specific regions for transcription or replication – these regions must be recognizable
importance of histone N-terminal tails
- enable organization and compaction of DNA
histone N-terminal tails
N-termini protrude out from the core particle of the histone and are less ordered - the tails are flexible so they are mostly disordered
they exit through the DNA superhelix through channels formed by the alignment of minor groove
H1 vs N-terminal tails
H1 is not required for forming the 30 nm filament, but tails are required - meaning the tails provide important nucleosome-nucleosome contacts needed
DNA double helix
a polynucleotide with a specific sequence of deoxyribonucleotide units covalently joined through 3’,5’-phosphodiester bonds; serves as the carrier of genetic information
histone
the family of basic proteins that associate tightly with DNA in the chromosomes of all eukaryotic cells and help condense DNA
chromatin
a thread-like structure, consisting of DNA and histones
nucleosome
the basic structural unit of chromosomes
30 nm filament
a condensed version of the nucleosome chain that forms upon binding of the histone H1
mitotic chromosome
a single large DNA molecule containing a discrete part of the genome-of an organism
essential cellular processes that rely on the modification of chromosomes
- Regulation of gene expression (ex: which genes are actively being expressed in a specific cell)
- DNA replication
- DNA editing and repair
- Recombination events
- The preservation of epigenetic tags (a chemical modification that occurs on DNA or specific amino acids in the histone proteins that DNA is wrapped around)
the nucleosome and DNA accessibility
Nucleosomes control accessibility of DNA to specific proteins, like transcriptional activators or repressors, therefore have a large influence on which genes are actively expressed
Nucleosome arrangements that are more open allow transcription, and closed arrangements repress transcriptions
classes of enzymes that regulate chromosome structure
- chromatin remodeling complexes
- histone modifying enzymes
chromatin remodeling complexes
they open DNA binding sites to allow binding of transcription factors
main functions of chromatin remodeling complexes
- repositions (slide) the nucleosome to a different location along the DNA strand
- eject the nucleosome from the DNA
- replace the nucleosome with the one that contains a histone variant - variant histone subunits impart special properties to the chromatin
different complexes for chromatin remodeling complexes
some are preferentially bound, resulting in physical movement - block or expose promoters = repressing or activating transcription OR eject or replace histones
some open up chromatin for gene expression - different ones have different mechanisms of action - some mobilize nucleosomes by forming a DNA loop – causes the nucleosome to slide to a new segment of DNA, enhancing DNA accessibility possibly (exposing a promoter that was previously blocked by the nucleosome)
histone modifying enzymes
covalently modifies the N-terminal tails of the histone proteins
heritable
cis acting modifications: Modifies the histone molecule directly - May result in opening or closing of the chromatin by tightening or loosening the arrangement of nucleosomes along the DNA
trans acting modifications: Involves other intermediary molecules - Attracts other proteins like transcription factors or chromatin remodeling factors which produce the chromatin change
which histone subunits have variants that alter DNA-binding affinity?
H2A and H3 have variants that differ in their N- and C- terminal sequences that confer special properties to the chromatin structure
H2A variants
H2AX, H2AZ, macroH2A - mainly differ in the C- terminal tail region which can recruit various proteins to the nucleosome
H2AX
DNA repair and genetic recombination
Becomes phosphorylated at Ser139 in the C-terminal region when a double-strand break occurs, attracting DNA repair proteins
If this phosphorylation is blocked, formation of the protein complex for DNA repair is inhibited
H2AZ
Associated with nucleosomes located at actively transcribed genes
Stabilizes the open state of chromatin, facilitating access of the transcriptional machinery to DNA in actively transcribed regions
MacroH2A
Abnormally large and contains a unique C-terminal domain
Involved in X chromosome inactivation
Shutting down one of the 2 X chromosomes in female mammals
H3 variants
H3.3 and CENPA = difference is the susceptibility of residues in the N-terminal tail to modifications such as methylation and phosphorylation
H3.3
During histone substitutions where active gene expression is occurring
Stabilizes the open state of chromatin, facilitating access of the transcriptional machinery to DNA in actively transcribed regions
CENPA
A H3 variant
Associated with repeated DNA sequences in centromeres
Contains a large extension that connects to the kinetochore (the site where spindle fibers attach and pull chromosomes apart during cell division)
chemical modifications of histones
- Acetylation of lysine
- Methylation of lysine and arginine
- Phosphorylation of serine
- Ubiquitination of lysine
are methylation and acetylation of histone tails reversible
yes
specific classes of enzymes that can add or remove histone tails
- HDAC’s (histone deacetylases)
- Remove the acetyl groups from histones - HATs (histone acetyltransferases)
- Add acetyl groups to histones - HMTs (histone methyltransferases)
- Add methyl groups to histones - Jumonji Family (KDMs, histone demethylases)
- Remove methyl groups from histones
specific covalent modifications
- methylation of arg residues
- acetylation of lysine
- methylation of lys residues
- phosphorylation
methylation of arg residues
Arginine can be methylated to methylarginine or 2 forms of dimethylarginine; double methylation can result in 1 methyl on each nitrogen of the guanidinium group or 2 methyl’s on one of the nitrogen atoms of the guanidinium group
acetylation of lysine
Performed by HATs
Acetylate specific residues in a histone tail, neutralizing the positive charge
HDACs will remove acetyl groups
Deacetylation of Lys residues result in transcriptional repression
methylation of lys reisudes
Methylated to monomethyl, dimethyl, trimethyllysinne
Lys9 or Lys14 of H3 can either be methylated or acetylated
phosphorylation
Commonly found on histone tails of H3 and H4
Can only occur on Ser, Thr, or Tyr residues because they have a hydroxyl group
Adds a negative charge to the tail
3 ways to alter chromatin structure/accessibility by modifying the nucleosome
- Chromatin remodeling complexes
- Reposition, eject or replace a nucleosome on the DNA strand - Variant histones that replace core histones within the nucleosome, influencing the chromatin structure
- Chemical modifications by histone modifying enzymes to the histone tails
- Acetylation or methylation, which alters chromatin structure
chromatin immunoprecipitation (ChIP) steps
- Cells are treated with formaldehyde to covalently crosslink nucleosomes to DNA. The cells are then disrupted, and genomic DNA is digested with micrococcal nuclease (an endo-exonuclease that preferentially digests single-stranded nucleic acids)
- An antibody to a specific modified histone is then used to immunoprecipitate the nucleosome-DNA complex. Any DNA not bound to a histone is washed away
- Protein-DNA crosslinks are reversed by heating and the released DNA is analyzed. If associations with a specific segment of the genome are suspected, DNA from this region can be amplified/quantified by PCR or qPCR. Otherwise, the released DNA can be sequenced (ChIP-Seq)
- Alternatively, the released DNA is labeled and used to probe a microarray. The pattern of hybridization on the array reveals the DNA sequences that associate with the nucleosomes (ChIP-Chip)
chromatin immunoprecipitation (ChIP)
technique used to determine the specific interactions between DNA and a protein, like a transcription factor
determines where on DNA sequence a protein exactly binds to
how do chromatin states get stabilized
- bromodomains
- chromodomains
bromodomains as enzymes to stabilize chromatin states
Recognize the acetylated Lys residues
Usually contained within a larger, multiprotein complex like the chromatin remodeling complex
If a chromatin remodeling complex contains a subunit with both a bromodomain and histone acetylase activity, the complex binds to an acetylated nucleosome so that a specific pattern of acetylation can be propagated in a targeted area of the chromosome
Leads to higher levels of gene expression
chromodomains as enzymes to stabilize chromatin states
Proteins that bind to methylated Lys residues
Often found in complexes with other enzymes that further modify chromatin structure
open vs closed chromatin states
- Acetylated nucleosomes are recognized by bromodomain proteins that may help stabilize the open chromatin state
- Methylated histones are recognized by chromodomain proteins that may help promote the closed state
epigenetic inheritance
the study of heritable changes in gene function that do not involve changes in the DNA sequence
where can epigenetic inheritance be transferred to
- from parent cells to daughter cells during division
- intergenerationally (between generations) between organisms from parents to their offspring
propagation of histone modifications
- epigenetic modifications during development
- controlling epigenetic modifications
- epigenetic modifications
H3-H4 tetramers during replication
remain bound to the DNA, unlike histone octamers that split apart during replication
marked histones during replication steps
- H3-H4 are randomly distributed on the 2 new daughter DNA duplexes made during replication, and coat only half of the total DNA after replication
- The new H3-H4 heterotetramers that lack the modification pattern of those they will replace are assembled onto the replicated DNA by the CAF-1 chaperone protein
- Parental H2A-H2B dimers remain in vicinity after being displaced by the replication fork and quickly reassemble with H3-H4 heterotetramers onto the newly replicated DNA, chaperoned by NAP-1. New, unmodified H2A-H2B dimers must be assembled on the newly replicated DNA
- 4 types of nucleosomes form on the daughter DNA strands:
- Old/parental H3-H4 and new H2A-H2B
- New H3-H4 and old H2A-H2B
- Entirely parental of H2A-H2B-H3-H4 histones
- Entirely new H2A-H2B-H3-H4 histones - The newly replicated DNA has only half of the parental epigenetic information on its histones, but the daughter DNA duplexes are “salted” with the parental histone modification pattern
