Lecture 4: Histone Modifications, Variants, and Chromosome Evolution
Histone Modifications, Histone Variants, and Chromosome Evolution
Variants:
One gene can encode several different products due to splicing. This is a crucial mechanism for increasing protein diversity from a limited number of genes.
Post-splicing, one gene can give rise to a multitude of different products (different mRNAs resulting in different proteins). This allows for tissue-specific or condition-specific protein expression.
At the genetic and RNA level, these are called variants. Variants include different mRNA transcripts arising from alternative splicing.
Once mRNAs are translated to protein, they are called isoforms. These are different versions of a protein that have slightly different functions or properties.
Recommended reading: Molecular Biology of the Cell, 6th edition (pages 175-236) or 7th edition (pages 206-228). These pages provide comprehensive details on gene expression and protein diversity.
Concepts
Understanding post-translational histone modifications. Post-translational modifications are key regulators of gene expression and chromatin structure.
Describing the function of histone variants. Histone variants contribute to chromatin diversity and have specialized roles in genome function.
Understanding components of centromeric chromatin. Centromeric chromatin is essential for accurate chromosome segregation during cell division.
Understanding the Y chromosome and its evolution. The Y chromosome has a unique evolutionary history, marked by gene loss and the acquisition of male-specific functions.
Post-Translational Histone Modifications
Histone proteins have tails, which are modified by adding different types of groups. These modifications alter chromatin structure and affect gene expression.
Post-translational modifications: covalent additions to the molecule after the protein is made. These include methylation, acetylation, phosphorylation, and ubiquitylation.
Covalent additions: phosphate groups, methyl groups, acetyl groups, etc. Each modification has a distinct effect on chromatin.
These modifications are a language for the cell. They communicate signals that regulate gene expression, DNA repair, and chromosome condensation.
Centromeric Chromatin
During mitosis, sister chromatids are separated at the centromere. The centromere is a specialized region of the chromosome that mediates chromosome segregation.
Microtubules are involved in pulling the molecules apart. Microtubules attach to the kinetochore, a protein complex assembled at the centromere.
Motor proteins (dynein and kinesin) play a crucial role in ensuring separation. They generate the force required to move chromosomes along microtubules.
Y Chromosome Evolution
The Y chromosome was once the same size as the X chromosome. Over millions of years, it has undergone significant reduction in size and gene content.
Due to mutations, it became a third of the X chromosome. These mutations led to the loss of genes and the accumulation of repetitive sequences.
Covalent Modifications on Core Histones
Core histones: histone 2A, histone 2B, histone 3, and histone 4. These proteins form the protein component of the nucleosome.
Each histone has variants. Histone variants have specialized functions and can alter chromatin structure.
N-terminal tails are modified by:-
Acetylation: Addition of an acetyl group, typically associated with increased gene expression.
Mono-, di-, tri-methylation: Addition of one, two, or three methyl groups, which can either activate or repress gene expression depending on the specific amino acid.
Phosphorylation: Addition of a phosphate group, often involved in cell signaling and chromosome condensation.
Ubiquitylation: Addition of ubiquitin, which can target proteins for degradation or alter their function.
Glycosylation: Addition of a sugar molecule, which can affect protein folding and interactions.
Modifications are reversible. This allows for dynamic regulation of chromatin structure and gene expression.
Histones help functionalize DNA for gene expression or repression. They play a critical role in determining whether a gene is transcribed or silenced.
Reversing Histone Modifications
Histone modifications are dynamic. They are constantly being added and removed to respond to cellular signals.
Acetyl groups are added by histone acetyltransferases (HATs), adding a negative charge to histone tails. HATs promote an open chromatin state and increase gene expression.
Acetyl groups are removed by histone deacetylases (HDACs), removing the negative charge. HDACs promote a closed chromatin state and decrease gene expression.
Methyl groups are added by methyltransferases or removed by histone demethylases. These enzymes play a crucial role in regulating gene expression and silencing.
Methyl groups have a slightly positive charge.
Review of Histone Tails
Histone tails are at the beginning of the histone protein. These tails are the primary site of post-translational modifications.
Amino acids on histones are named by indicating:-
Which histone it is (e.g., histone 2A).
The amino acid (e.g., lysine 15).
The kind of modification it has received: This nomenclature allows for precise identification of histone modifications.
Amino acids that can be phosphorylated: serine, threonine, and tyrosine. These amino acids have hydroxyl groups that can be phosphorylated.
Lysines can be acetylated and methylated. These modifications play critical roles in regulating gene expression.
A lysine can either be methylated or acetylated but not both simultaneously. This mutually exclusive modification affects chromatin structure and gene expression.
Histone 3
Histone 3 plays a significant role in the cell cycle. It is involved in regulating chromosome condensation and segregation.
It indicates when chromosomes are in interphase and when the cell is ready to enter synthesis and mitosis. Specific modifications on histone 3 signal the cell's progress through the cell cycle.
Different variants of histone 3 play a role in signaling the cell's stage in the cell cycle. Histone 3 variants like CENPA are essential for centromere function.
Modifications are added to the side chains of amino acids. These modifications alter the properties of the histone tails and affect their interactions with other proteins.
The Cell's Language
The cell communicates through proteins, DNA, nucleic acids, and hormones. These molecules transmit signals that regulate cellular processes.
Proteins are like letters, several proteins make up a word, and several words make up a sentence (a command). This analogy helps to understand the complexity of cellular communication.
The cell communicates with itself and surrounding cells through proteins and other molecules. This communication is essential for coordinating cellular activities and maintaining tissue homeostasis.
We are trying to interpret this language, particularly the language of post-translational modifications on histones. Understanding these modifications will provide insights into gene regulation and disease.
Histone Core
DNA wraps around the histone core 1.5 times. This packaging helps to condense the DNA and regulate gene expression.
Not just histone tails have post-translational modifications; the core of the protein also has modifications. These modifications can affect histone-DNA interactions and chromatin stability.
Modifications in the core include acetylation, methylation, phosphorylation, ubiquitination, etc. While less studied than tail modifications, they are increasingly recognized for their importance.
We understand the role of histone tails better than the modifications within the core of the histone protein. Research is ongoing to elucidate the functions of core histone modifications.
Histone Code Hypothesis
There are numerous possible markings on individual nucleosomes. This combinatorial diversity allows for fine-tuned regulation of gene expression.
Nucleosome diversity is expanded by histone variants. These variants contribute to the functional specialization of chromatin.
Covalent modifications and histone variants have specific meanings for the cell. They act as signals that direct various cellular processes.
The histone code hypothesis suggests that histones instruct the cell about:-
Transcription: Determining whether a gene is actively transcribed or silenced.
DNA damage: Signaling the presence of DNA damage.
Repair: Recruiting DNA repair machinery to sites of damage.
Expression and suppression: Regulating the expression of genes.
Acetylation is a signal for expression of a gene on a histone tail. It promotes an open chromatin state that allows for transcription.
Methylation is a signal for suppression. It can lead to chromatin condensation and gene silencing.
Histone variants can indicate repair and damage. They can recruit specific repair proteins to damaged DNA.
How Histone Modifications Are Used in Regulation
Histone tails can signal machinery to wind the DNA, hypercoil it, make it heterochromatic, or expand that region. These structural changes affect gene accessibility and expression.
This information comes from histone tail post-translational modifications. The modifications are recognized by specific protein complexes.
A code reader complex reads the histone tail post-translational modifications. These complexes bind to specific modifications and recruit other proteins.
The complex is made up of different proteins and has regions to read histone tail modifications. Each protein domain recognizes a specific modification.
The complex recruits the appropriate proteins due to conformational changes. This allows for the recruitment of specific enzymes and regulatory factors.
This can result in reading and propagating a signal or making additional modifications. The reader-writer complex can spread modifications along the chromosome.
Biological Outcome of Histone Modifications
Heterochromatin formation or spreading across a chromosome, usually resulting in gene silencing. Heterochromatin is a condensed form of chromatin that is typically associated with transcriptional repression.
A combination of marks is important (e.g., methylation of lysine 4 and acetylation of lysine 9 equals gene expression). The combination of different modifications creates a specific code that determines the outcome.
Serine 10 phosphorylation and lysine 14 acetylation also equal gene expression. These modifications are often associated with immediate early genes.
Lysine 27 methylation is often found in the inactivated X chromosome, which is hypermethylated and heterochromatic. This modification is crucial for X chromosome inactivation in females.
Hox Genes
Important genes that must be silenced for the majority of the life of a multicellular organism. These genes control body plan development.
Encode transcription factors that are morphogens. Morphogens are signaling molecules that determine cell fate based on their concentration.
Induce different types of genes to be expressed throughout the body at different concentrations. This precise regulation is essential for proper development.
Reader Writer Complex
Can spread heterochromatin by reading and propagating the signal to add marks to histone tails. This complex can create large domains of silenced chromatin.
Can dissociate heterochromatin into euchromatin. It can also remodel chromatin to allow for gene expression.
Interacts with a barrier protein that binds to a barrier sequence. Barrier proteins prevent the spread of heterochromatin.
If the barrier protein or sequence is lost, heterochromatin can spread further, and genes that should be expressed are silenced. This can lead to aberrant gene expression and disease.
Barrier Protein
Highly attached to the nuclear pore when adjacent to regions that are highly heterochromatic. This localization helps to prevent the spread of heterochromatin.
Physically blocks the spreading of heterochromatin. It acts as a physical barrier to the reader-writer complex.
Protects and encases other regions so the reader-writer complex cannot continue to write. This prevents the silencing of genes that should be expressed.
Has enzymatic activity and can physically inactivate the reader-writer complex. This provides an additional layer of control over heterochromatin spreading.
Histone Variants
The reader-writer complex reads histones, histone tails, and specific histone variants. Histone variants contribute to the diversity of chromatin and its functions.
Histone Variant 3
Histone 3 gene can encode for three different histone 3 proteins: histone 3.3 and CENPA. These variants have distinct functions within the cell.
Encoded by the same gene but have different regions, contributing to differences in function. Alternative splicing and post-translational modifications contribute to the diversity of histone 3 variants.
The structure of a protein contributes to its function. The unique structure of each histone variant allows it to perform a specific function.
Function of Histone Variants
Histone 3.3: When placed within a chromosome, that region is specifically for transcriptional activation. Histone 3.3 is associated with actively transcribed genes.
CENPA: When part of the nucleosome, encodes the information that that region is designated as a centromere. CENPA is essential for centromere identity and function.
Centromere: The region that has to collect as heterochromatin to pull sister chromatids apart during mitosis. The centromere is a specialized region of the chromosome that is required for accurate chromosome segregation.
Other Histone Variants
Histone 2A, histone 2AX, histone 2AZ, macro histone 2A: all result in something different when incorporated into a nucleosome in the genome. These variants have specialized functions in DNA repair, transcription, and chromosome structure.
The addition includes histone chaperones that swap them with a nucleosome core and ATP-dependent chromatin remodeling complexes. These factors facilitate the incorporation of histone variants into chromatin.
Histone cores can be swapped in and out, conveying different messages. This dynamic exchange allows for rapid changes in chromatin structure and function.
Centromeric Protein A (CENPA)
Conserved in various species (C. elegans, yeast, flies, etc.). CENPA is an evolutionarily conserved protein that is essential for centromere function.
Indicates the centromeric region of the chromosome (centromere). CENPA defines the location of the centromere on the chromosome.
Mutations in CENPA can lead to CREST syndrome (an autoimmune disease). This highlights the importance of CENPA in maintaining genome stability and preventing autoimmunity.
Autoimmune disorder that targets CENPA leads to lesions on the body and intestinal issues. The autoimmune response against CENPA can have severe consequences for human health.
Treatment tempers the autoimmune disorder but is a lifelong disease. There is currently no cure for CREST syndrome, and treatment focuses on managing the symptoms.
Mitotic Chromosomes
Two sister chromatids with centromeres. Each sister chromatid contains a centromere that is essential for chromosome segregation.
CENPA is found in these regions. CENPA is specifically localized to the centromere.
During interphase, CENPA is punctate and found in many different regions due to chromosome territories. Chromosome territories are distinct regions within the nucleus where chromosomes are located.
Localization of CENPA
90% of the centromere has CENPA, and 10% has histone 3. CENPA is the major histone variant found at the centromere.
Immunofluorescence shows that they don't always overlap. This suggests that CENPA and histone 3 may occupy different regions within the centromere.
Fluorescence can mask the resolution, making it difficult to distinguish between the two. Higher resolution techniques are needed to fully understand the organization of the centromere.
Electron microscopes and antibodies conjugated to metals provide higher resolution. These techniques allow for visualization of the centromere at the nanometer scale.
Structure of Chromosome
CENPA-containing nucleosomes stick out on the very edge of the sister chromatid. This positioning facilitates the attachment of the kinetochore.
Kinetochore attaches to the CENPA-containing nucleosomes to pull sister chromatids apart. The kinetochore is a protein complex that links the centromere to microtubules.
Histone 3-containing nucleosomes are clustered in the core of the centromere, not on the side. This suggests that histone 3 may play a structural role within the centromere.
Review of Centromere
Sister chromatids are attached at the centromere. The centromere is the point of attachment between sister chromatids.
Kinetochore assembly line recognizes the centromere to separate the sister chromatids. The kinetochore is responsible for attaching the chromosomes to microtubules and ensuring their proper segregation.
Centromeric heterochromatin is not part of the region that will be pulled. The heterochromatin surrounding the centromere is important for its structural integrity.
Loading of CENPA
Before S phase, CENPA is at 100%. CENPA is fully loaded onto the centromere before DNA replication begins.
During S phase, the two sister chromatids enter G2 phase with 50% of CENPA loaded. CENPA is diluted during DNA replication.
Once sister chromatids are pulled apart and enter a new cell at G1 phase, CENPA is reloaded to 100%. CENPA levels are restored after cell division.
HGRP
HGRP loads CENPA (histone 3 variant) in late telophase or G1. HGRP is a key regulator of CENPA loading.
Named Holiday Junction Recognition Protein but doesn't necessarily play a role during holiday junction formation. The name is misleading, as HGRP's primary function is CENPA loading.
In the absence of HGRP, CENPA is just everywhere in the nucleus because it's not loaded to the centromeric DNA. HGRP is required for the proper localization of CENPA to the centromere.
Chromosome Evolution: Evolution of the Y Chromosome
The Y chromosome used to look like an autosomal pair (like identical twins). The Y chromosome originated from an autosome.
Currently, the Y chromosome is approximately one-third of the size and recombines only at the tips with the X chromosome. The Y chromosome has undergone significant reduction in size and gene content.
Autosomes recombine along the entire length of the chromosome. Recombination is the exchange of genetic material between chromosomes.
Origin of Y Chromosome
Sex chromosomes began as autosomes and were called proto-X and proto-Y 300,000,000 years ago. The sex chromosomes evolved from a pair of autosomes.
Structural changes in the Y chromosome resulted in the modern form we recognize today (one-third of the X chromosome). These changes included inversions, deletions, and gene loss.
Proto-X and proto-Y recombined during meiosis. Meiosis is a type of cell division that produces gametes (sperm and egg cells).
Both the X and Y chromosome contained a SOX3 gene. SOX3 is a transcription factor involved in neural development.
In the Y chromosome, the SOX3 gene acquired a mutation that converted it into SRY (male determination). SRY is the master sex-determining gene on the Y chromosome.
RPS4 is found on both the Y and X chromosome and has retained similar functions. RPS4 is a ribosomal protein that is essential for protein synthesis.
History of Y Chromosome
Y chromosome recombined with itself, forming a large inversion. This inversion prevented recombination between the X and Y chromosome.
The Y and X chromosome were no longer able to recombine with one another along that length. This lack of recombination led to the degeneration of the Y chromosome.
The proto Y now is losing some regions because it couldn't recombine with the X. The Y chromosome has lost many genes over millions of years.
The Y chromosome is now only capable of recombination at the tips. This limited recombination occurs in the pseudoautosomal regions.
Due to this inability to recombine, the proto Y accumulated mutations, therefore, becoming susceptible to deletions. Mutations and deletions have contributed to the reduction in size and gene content of the Y chromosome.
X Chromosome and Sustainment of it's Size
Recombination helps repair when genes are completely lost.
*Whenever a male gave rise to a female, that x chromosome that the male had in the female now that x chromosome was sustained due to the other x chromosome that that female inherited from their mother from the maternal side.
X Chromosome
It is lethal to not have an x chromosome.
As you can understand, it's because the y chromosome is missing several genes that normally would have supporting for life itself.
Saving Grace to the Y Chromosome
*The y chromosome acquired an autosomal DAS gene, relocated to this y chromosome, and the DAS gene was copied multiple times onto the y chromosome.
*Due to DAS being copied multiple times results to palindromic sequences.
*The y chromosome now is stable.
Million Year Old Palindromes
*Called the genomic wastelanders.
*Contain stetches of palindromic DNA that stretch on both sides along the length the Y chromosome.
*Hypothesized that these palindromes allow for genes that have developed mutations to recombine with normal genes for repair onto the Y chromosome.
*Results in a structure of