Acquired inheritance

An idea used by Darwin to explain heritable variation. Put forth by Lamarck. Gemmules respond in adaptive ways to an individual's external environment and experience. Altered gemmules are passed on to offspring, allowing for inheritance of acquired characteristics. Today it is considered incorrect, disproved by experiments of Joshua and Esther Lederberger as well as Luria Delbruck.

Anti-silencing factor

A histone chaperone which disassembles the nucleosome right before passage of the replication fork. Bound to the helicase MCM. It breaks the interaction between H3/H4 dimers.

Chromatin assembly factor

A histone chaperone. Studied in Cheloufi et al (2015). Travels right behind the replication fork, associating with the replicative clamp PCNA. Old histones are ferried to it by ASF1 and FACT, and it re-assembles them into nucleosomes. If mutated, reduces frequency of conversions at the telomere. A model for how heterochromatin can reversibly activate nearby genes (causing epigenetic changes during PEV and TPE): new histones are also delivered to CAF1, and assembled in the nucleosomes. If the replication fork stalls, the supply of old histones is disrupted; it is possible that CAF1 and other chaperones continue to assemble nucleosomes from new histones only, losing epigenetic marks. It is suppressed in homozygous and heterozygous MEFs that carry certain alleles. Its suppression enhances reprogramming for different cell conversion systems, by multiple magnitudes, in a variety of cell types, depending on dose and duration. Suppression causes changes in histone modifications, resulting in a more open chromatin structure at enhancer elements during early reprogramming. HPS cells were reprogrammed in the presence of the suppression of CAF1 components and similar enhanced reprogramming results were obtained. Acts as a gatekeeper across different cell types, even when using transcription factors other than OKSM to induce cell lineage transformation. 

Molecular biology shows that genetic information is faithfully transmitted, from DNA, to RNA, to protein. It can go backwards, with RNA-mediated repression of genes.

DNA ↔ RNA → protein

The histone chaperone CAF1 safeguards somatic cell identity

Ectopic expression of 4 OKSM transcription factors is enough to reprogram cells into iPSCs. Researchers sought to elucidate mechanisms in place to safeguard somatic cell identity. Changes in histone modifications are accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets, and activation of associated genes. Discoveries pave the way for further studies to establish enhanced strategies for the modulation of cellular plasticity in regenerative and research settings.

Methylation of the ribosomal RNA gene promoter is associated with aging and age-related decline

DNA methylation profiles of ribosomal RNA (rRNA) genes can be used as a biomarker for detecting onset of mammalian age-related diseases and cancers. Methylation of critical housekeeping regions, such as those involved with gene transcription, impacts overall cell function. Hundreds of rRNA loci are silenced by epigenetic mechanisms, due to tendency to recombine in the genome. Studied the effect of methylation of an rRNA gene promoter on rRNA transcription levels across aging. rRNA levels decrease with age in both humans and rats. In humans, there was correlation between methylation of CpG_5 in the promoter and phenotypes of age-related decline. Lack of concordance between methylation and age indicates that profiles are not markers for chronological aging, but are for biological aging. There was correlation between methylation and aging in rat samples; a marker for both biological and chronological aging. This may be due to the more varied environmental conditions in human subjects compared to rat subjects. Emphasized that understanding epigenetic marks can lead to prevention of age-related diseases. Methylation is not necessarily causal to aging. Showed correlation only; direct evidence required to show how methylation leads to age-related decline. Future research may identify functional effects of methylation on rRNA levels and aging. Proposed model:

Methylation of rRNA gene promoter → increased silencing → lack of ribosome biogenesis → less protein synthesized for cellular function

ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference

Measured imitations switch (ISWI) chromatin remodeler activity in response to a diverse nucleosome library of 115 mutated and modified nucleosomes. Generated a large dataset of remodeling rate kinetics, giving insight into regulation of ISWI interactions. Chromatin remodelers interact with nucleosome post-translational modifications (PTMs), which modulate their activity, controlling gene expression. Found that PTMs generally have similar effect on all seven ISWI complexes analyzed, except NoRC. Each PTM either activates or inactivates all ISWI complexes. The ATPase SNF2h alone has distinct reaction, indicating that reader subunits play an important role in reaction to PTMs. NoRC has different response to PTMs, particularly acetylation marks. Found an association between ISWI complexes and an acidic patch on H2A and H2B histones. Nucleosome library and associated dataset produced represent groundwork upon which there is much potential for future studies on chromatin remodelers. Not an in vivo study; lacks native heterochromatin/euchromatin structure and other regulatory interactions. Uses complete ISWI complexes, which is an improvement from many previous studies which looked at single subunits.

A model for how old histones are disassembled, and their epigenetic marks transmitted during DNA replication. There is some uncertainty about the mechanism of transfer of histones at the replication fork.

1. Two chaperones, ASG1 and FACT, disassemble the nucleosome right before the passage of the replication fork.

2. ASG1 and FACT ferry the histones to a CAF1 right behind the replication fork. H2A/H2B are transferred as dimers. In most cases H3 and H4 are transferred as un-split tetramers, though there is some evidence that they are split into dimers.

3. New H3/H4 histones are brought to CAF1, which assemble into new and old histones; the mechanism is not firmly established. New histones are recognized by their predisposition marks.

4. Somehow modifications of old histones impose the same modifications on the new histones.

Enhancer of variegation

Suppressor genes of white^V in Drosophila. The fly's eyes are mostly white, with some red patches. It enhances the spread of heterochromatin. Homologues have been identified in all metazoans; key regulators of many epigenetic transactions. Mutated in cancers and various rare diseases. Encodes key readers, writers, and erasers in epigenetic marks. Characterization formed the foundation of modern epigenetics.

Facilitates chromatin transcription

A histone chaperone which disassembles the nucleosome right before the passage of the replication fork. Bound to the helicase MCM. It works with H2A and H2B.

Linker histone

A histone which is associated with linker DNA between nucleosomes. Without H1, nucleosomes are not disrupted, however the tight 30 nm chromatin fibre packaging is lost; it is just "beads on a string". Sits between the nucleosome and the linker DNA.

Nucleosome loss leads to global transcriptional up-regulation and genomic instability during yeast aging

Found that histone protein levels decrease in yeast as they age. Nucleosomes become less well positioned, or move to sequences predicted to better accommodate histone octamers. Altering these epigenetic changes may prevent onset of aging or age-related illnesses in humans. Transcriptional up-regulation of genes leads to shorter lifespan. Retrotransposon transcription increases, leading to greater genomic instability. dsDNA breaks allows mtDNA translocation in genomic DNA. Leads to transcriptional induction of all yeast genes, elevated levels of DNA breakage, mitochondrial DNA transfer to the nuclear genome, large-scale chromosomal alterations, translocations, and retrotransposition during aging. Overexpressing H3 and H4 extends lifespan.

Homo sapiens

Have 3,300 Mb of DNA, 20,500 genes, 23 chromosomes, and 9 genes/Mb. Have five times as many genes as E. coli. Lifecycle takes around 20 years to complete. There are around 10¹¹ cells in the body. Genome size is 6 x 10⁹ bp. There is a higher order of chromosome structure. Sex is complicated. Sex determination is with X and Y chromosomes.

Imitation switch chromatin remodeler

Protein complexes that catalyze ATP-dependent nucleosome sliding along DNA. Includes an ATPase domain (SNF2h), and "reader" subunits which interact with histone post-translational modifications.

Analysis of epigenetic stability and conversions in Saccharomyces cerevisiae reveals a novel role of CAF1 in position effect variegation

PEV phenotypes are characterized by the robust multigenerational repression  of a gene located at a certain locus (gene silencing) and occasional conversions to a fully active state. Addressed important aspects of PEV: frequency of conversions. Developed a model for various PEV scenarios based on various states of conversions. Assays revealed that histone chaperone CAF1 is involved in control of epigenetic conversions.

PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum

Variant antigen in erythrocyte membrane protein 1 (PtEMP1) is expressed on the surface of P. falciparum-infected red blood cells, and is a critical virulence factor for malaria. Each parasite has 60 antigenically distinct VAR genes, encoding a different immune-evasion mechanism. How 59 of the VAR genes are silenced is unknown. Knocking out SET gene controls H4K36Me3 on the VAR gene. Focused on determining the possible regulation mechanisms of var genes through epigenetic factors. Determined that PfSETvs is one mechanism that controls var gene silencing through H3K36Me. Discovered that PfSETvs knockout results in expression of all 60 var genes. Provides insight into mechanisms of PfSETvs ortholog gene repression in other eukaryotes. Parasites containing PfSETvs knockout may lead to development of malaria vaccine. RTS,S recombinant vaccine is currently being tested, and has a success rate of 26 - 50%. Conclusively, the study provided a new target for malaria therapy, and has provided a stepping stone for further research on the relationship between gene silencing and var gene regulation.

Six experiments were conducted:

1. Determining if any of the 10 PfHKMTs (PfSET genes) or 3 PfHKDMs contribute to var gene silencing.

2. Determining if multiple var genes can be transcribed at the same time.

3. Determining if var genes are able to translate multiple PfEMP1s on the surface of an infected red blood cell.

4. Determining the effect of PfSETvs knockout on methylation of P. falciparum genome, and identify a relationship between histone methylation and location of var genes.

5. Determine if the reduction of H3K36Me3 by PfSETvsΔ is specifically associated with activation of parasite clonally variant genes.

6. Determine the role of H3K36Me3 in var gene silencing in active vs. silent var genes.

Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes

Evaluated different epigenetic regulators for their ability to protect transgene expression at telomeres. Found that matrix attachment regions (MARs), ubiquitous chromatin opening elements (UCOE), and chicken cHS4 insulator acted as barrier elements.

Methyl-CpG binding domain

Supports the model that DNMT methylates using the old methylated strand as a guide for the enzymes. Binds to hemi-methylated DNA. It reads for CpG islands, and directs the activity of DNMT, copying methylation onto the other DNA strand. In Neurospora, it is NP95. Includes MBD1, MBD2, and MBD3, which are reader proteins that direct DNMT1 to the newly synthesized DNA strand. MBD2 and MBD3 are interchangeable subunits of NuRD nucleosome modeling complex; mutations in these impair cell differentiation and cause cancer.

Cancer-related epigenome changes associated with reprogramming to induced pluripotent stem cells

Ability to induce pluripotent stem cells from committed, somatic human cells provides tremendous potential for regenerative medicine. Neoplastic potential inherent to reprogramming must be understood; provides a model for tumorigenesis. Used genome-wide assays. Identified cancer-related epigenetic abnormalities which arise early during reprogramming and persist in induced pluripotent stem cell clones.

Origin recognition complex

Binds to ACS in mating type loci, and recruits SIR1, allowing for recruitment of SIR2.

Proliferating cell nuclear antigen

A replicative clamp which travels right behind the replication fork. Associates with CAF1 and DNMT. A "sliding clamp". It is often a mark of cancer, found in proliferating cells. A lot of proteins may bind to it, but it can only have three bound to it at a time. Regardless of how many factors are bound to it, it knows when to replicate.

H3K56Ac

A mark which is used to recognize new histones during DNA replication and epigenetic transmission. There is a reader which directs enzymes to modify the new, and not the old histones.

A six months exercise intervention influences the genome-wide DNA methylation pattern in human adipose tissue

The epigenome differs between cell types, characterized for a few human tissues. Environmental factors potentially alter the epigenome. Describes genome-wide patterns of DNA methylation in human adipose tissue, and concludes that exercise induces genome-wide changes, potentially affecting adipocyte metabolism.

Budding yeast

A single-celled eukaryote. Has 15 Mb of DNA, 6,200 genes, 17 chromosomes, and 400 genes/Mb. Genome size is 12 x 10⁶ bp. Heterochromatin has histone deacetylation (SIR2), no trimethylation of histone H3K9, trimethylation of H3K79, no DNA methylation, and no HP1. Not everything found in yeast applies to Metazoa. Used in the making of beer and bread. A model organism; became popular in research in the 1970s, especially in cancer research. Lifecycle takes 90 minutes to complete; very fast. Billions can live in a petri dish. There is higher order of chromosome structure. Has very simple sexual reproduction. Can be used to study gene silencing at mating loci. Sex determination (a or α mating type) is with four genes in two loci: MAT, HML, HMR, and HO. Can grow and divide as either diploid or haploid cells. Undergo standard eukaryotic cell cycle. Its stage in cell cycle can be determined under a microscope. In S phase, there is a bump on the cell surface, which becomes two cells during mitosis. Daughter cells may remain attached after mitosis. Sporulates during bad conditions, undergoing meiosis, in which four haploid cells burst from the cell: two each of a and α mating types. After budding, there is a scar on the cell, which can be used to determine the age of the cell. Disadvantages to studying PEV include no DNA methylation, and no HP1. Advantages include estimating frequency of epigenetic switches can be observed forwards and backwards. Its lack of DNA methylation can be good for epigenetic research, because it removes "background noise". We learned that genes regulate chromatin structure, with many writers, readers, and erasers. Proteins at the replication fork regulate epigenetic switching, especially when stalled.

Enforcement of a lifespan-sustaining distribution of SIR2 between telomeres, mating-type loci, and rDNA repeats by RIF1

Showed that RIF1 maintains replicative lifespan by stabilizing another age-related chromosomal domain, harbouring ribosomal DNA (rDNA) repeats. Siruin histone deacetylases, including SIR2 and mammalian SIRT6, operate at multiple age-related loci, and limiting their location to these loci can promote lifespan-sustaining roles elsewhere in the genome.

Something about silencing 2

SIR2 is a suppressor mutation of sir3. An H4K16 acetyltransferase (HAT); a writer of H4K16Ac. It is constantly competing with SIR2. A mutation reduces frequency of conversions at the telomere. A HAT that travels behind the replication fork. It travels with CAF1. Has an unknown relationship to replication.

FANCJ couples replication past natural fork barriers with maintenance of chromatin structure

Homozygous mutations in the helicase FANCJ cause Fanconi anemia. Replicative stress, caused by replication fork barriers, can negatively impact the epigenome. Researchers propose that FANCJ plays a crucial role in maintenance of genomic and epigenomic stability by facilitating replication or movement past DNA-structure mediated replication barriers. Stalled forks were replication competent and capable of restarting DNA synthesis upon removal of replicative stress. Guanine rich sequences were found in promoter regions of many tumour suppressor genes; changes in chromatin compaction may result in their silencing.

Switched off

Non-active genes. In differentiated eukaryotic cells, 60 - 70% of genes (in humans, 15,000 genes) are switched off In the same tissue, the pattern of gene expression is maintained through multiple cell divisions. Different sets of genes are silenced in different cell lineages. The silenced genes are very rarely unsilenced, which can lead to pathology such as cancer.

Suppressor of variegation

Suppressor genes of white^V (PEV) in Drosophila, found in a genetic screen. The fly's eyes are mostly red, with some white patches. It inhibits the spread of heterochromatin. Includes Su(var)2-5 and Su(var)3-9; both encode proteins related to the histone mark H3K9Me. Homologues have been identified in all Metazoans; key regulators of many epigenetic transactions. Mutated in cancers and various rare diseases. Encodes key readers, writers, and erasers in epigenetic marks. Characterization formed the foundation of modern epigenetics.

Subtelomeric region

DNA sequences adjacent to the telomeres. Contain histones which are highly condensed into heterochromatin. In east, there are short TGGG (TG₃) repeats, as well as ACS and Abf1 sites. There are 1,000 repeats, scattered in the subtelomere. Repetitive and conserved, but not as conserved as the telomeres. Genes are silenced in yeast. Genes can convert their state (silent vs. active) about once every 20 divisions: PEV and TPV. There is variegation. Allows cells in the population to express genes based on the environment. In yeast, most transposons are found in the subtelomere.

A substitution mutation is inserted into H3 and H4 histones to perform mechanistic studies on histone modifications. For example, a K → Q mutation mimics acetylation of lysine residues. Can be used to look at gene silencing function in yeast. Identifies proteins that act through positions of the histones when H4K16 is mutated to Q. The phenotype, due to loss of silencing, is observed. The whole genome of H4K16Q mutant cells is mutagenized to find cells with restored silencing using a genetic screen; this is the suppressor mutation. Identifies SIRs and RAP1 as proteins needed to silence mating type loci in yeast, if silencing is impaired.

1. Create a mutation that disrupts a function.

2. Produce a cell culture with mutant cells.

3. Treat this culture with a mutagen so that every cell contains about one additional mutation in any gene.

4. Identify a cell that has regained the function; this cell has a secondary suppressor mutation, which suppresses the effect of the first mutation. If gene silencing is impaired, the yeast cells will express ADE3 gene, and form white colonies. Look for rare red colonies which suppress the defect, and ADE2 gene is silenced.

5. Identify the gene that carries the secondary mutation. Cells are transformed with a library of plasmids, each expressing one gene. Each cell normally acquires one plasmid. Isolate the plasmid and sequence the gene.

A model for how epigenetic marks transfer to new histones. All steps are highly speculative.

1. Reading and copying of histone code. Epigenetic marks (H3K9Me or H4K16A) recruit a specific dedicated reader/enzyme pair. The reader and writer may be separate complexes, the reader directing the enzyme's work, which would otherwise be random. Concomitant with this step is the recognition of the new histones by their predisposition mark (H3K56Ac); a reader directs enzymes to modify only new histones.

2. The enzyme catalyzes the same modification on the residue of the new histone.

3. The histone code is in action. H4K16Ac and H3K56Ac are recognized by another enzyme, which catalyzes mon-methylation of K3K9.

4. The predisposition mark is removed. The histone is no longer "new", and is recognized as carrying the same histone code as the hold histones.

Variable surface glycoprotein

Genes found in trypanosome blood parasites. Coat the parasite, and are exposed to the immune system. There are about 1,000 VSGs, even though it only uses 50 - 60. Most are subtelomeric genes, silenced and variegated by a TPE mechanism similar to that of yeast. While the immune response to VSGx builds up, the parasite has an epigenetic switch to another VSG. Subtelomeres contain assemblies of proto-silencers, anti-silencers, and insulators that compromise a sophisticated and seemingly indestructible switching mechanism. If we suppress switching, the immune system will overwhelm the parasites.

Corn

A plant. Has around 3,500 Mb of DNA, around 50,000 genes, and 10 chromosomes. Can have variegation where kernels have identical genotypes, but one is solid colour and the other is spotted.