Advanced Concepts in Genetics: Quiz 2 – Flashcards

Activator

A locus in corn discovered by Barbara McClintock. Controls chromosome 9S dissociation at Ds. A transposon containing a transposase gene.

Chromatin immunoprecipitation

A weak interaction between proteins and DNA in assay is captured by formaldehyde in vivo. The DNA is broken into pieces, and protein is filtered out, with DNA still attached, which can then be sequenced.

Maize

A model organism.

DNA methyltransferase 1

Performs de novo DNA methylation. A mutation in mice caused removal of DNA methylation of the paternal H19 allele, which resembles the maternal allele.

Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes

The pluripotent state of embryonic stem cells (ESC) is produced by active transcription of genes that control cell identity, and repression of genes encoding lineage-specifying developmental regulators. ESC cohesion ChIA-PET is used to identify local chromosomal structures at active and repressed genes. Produced a map of enhancer-promoter interactions, revealing that super-enhancer genes generally occur within chromosome structures formed by looping of two interacting CTCF sites, co-occupied by cohesion. Loop structures form insulated neighborhoods, whose integrity is important for expression of local genes; encodes lineage-specifying developmental regulators. Looked at chromatin structures that ultimately affected ESC production and maintenance. Stem cells were produced and maintained by actively transcribing genes that control cell identity while genes encoding lineage-specifying developmental regulators are repressed. 

Dissociation

A locus in corn studied by Barbara McClintock. The dominant C, Sh, and Wx alleles on breakable chromosome 9 are always lost together. The chromosome always breaks at the same locus: Ds. A second locus controls chromosome 9S dissociation at Ds: Ac. Ds is itself a defective Ac element which requires an intact Ac for transposition. Deletion of the whole arm of 9S at Ds removes C, Sh, and Wx.

Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells

Focuses on endogenous retroviral elements (ERV) silencing in late mouse embryonic stem cells, maintained by H3KMe3. Revealed the importance of H3.3 in silencing ERVs. Proposed a model for a positive feedback loop that maintains heterochromatic state. Found experimental evidence to support the role of H3.3 in maintaining H3K9Me3 at ERVs. Researchers can further their understanding of how heterochromatin is maintained in ESCs, maintaining genome integrity in developing embryos. Critical in developing therapeutic treatments in oncology and autoimmune disorders. Proposed model:

1. KAP1 recognizes ERVs through sequence-specific KRAB zinc finger DNA binding proteins.

2. KAP1 recruits ATRX-DAXX independently of its interaction with ESET.

3. ATRX-DAXX deposits H3.3 at IAPs, facilitating efficient KAP1 association with chromatin.

4. ESET methyltransferase is recruited, resulting in H3K9Me3-mediated silencing of ERVs.

Fluorescence in situ hybridization

A method of using fluorescent probes to visualize RNA. Can be used to see Xist transcripts on the X chromosome.

Disruption of DNA-methylation-dependent long gene repression in Rett syndrome

Disruption of the MeCP2 gene leads to Rett syndrome (RTT). MeCP2 encodes a methyl-DNA binding protein that has been proposed to function as a transcriptional repressor. Despite numerous mouse studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how it regulates transcription. Present evidence that MeCP2 represses gene expression by  binding to methylated CA sits within long genes, and that in neurons lacking MeCP2, decreasing expression of long genes attenuates RTT-associated cellular deficits. Focuses on epigenetic mechanisms beyond neurodevelopmental disorders, specifically transcriptional repression through DNA methylation. Looked at the role of MeCP2, which was previously proposed to play a role in transcriptional regulation. Proposes a model by which MeCP2 represses transcription in a length-dependent manner in neuronal tissue. Contributes to the broad field of epigenetics.

Immunodeficiency centromere instability facial anomalies

A human genetic disease syndrome linked to DNA methylation aberration. A rare disorder. Inherited as a recessive trait. Branched chromosomes form in cultured lymphocytes. Caused by mutations in the gene encoding de novo DNA methyltransferase, Dnmt3B. Causes loss of DNA methylation in the satellite DNA around the heterochromatic centromeres on chromosomes 1, 9, and 16. This gene is involved with parental imprinting.

Insulin-like growth factor 2

Regulates embryonic growth. A classical example of reciprocally imprinted genes. Occupies a locus near H19 on chromosome 11, 11p15. In any diploid somatic cell, only the paternal Igf2 is expressed, and the maternal is always silenced. Controlled by a methylation-sensitive chromatin boundary, ICR. Paternally expressed. Mutant mice are 40% smaller than wild-type; this supports the parent offspring conflict hypothesis, because the mother "wins", and the parasitic embryo loses. Studied in Zhang et al (2017). Loss of expression and biallelic expression is characteristic of many human tumours. Molecular mechanisms responsible for reactivation of normally suppressed maternal alleles in tumour cells is unclear. Its promoter complex includes an interaction with miR482 and P2, involved with its reactivation in the maternal allele.

Insulin-like growth factor 2 receptor

An antagonist of Igf2. It is maternally expressed. Mutant mice are oversized, and inviable; this supports the parasitic offspring conflict hypothesis, because the father wins, not the selfish mother. However, the child dies.

Translating dosage compensation to trisomy 21

Tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, inserted a large, inducible XIST transgene into DYRK1A locus on chromosome 21 in Down's syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing, and DNA methylation, to form a "chromosome 21 Barr body".

Adaptation to P element transposon invasion in Drosophila melanogaster

PiWi-interacting RNAs (piRNAs) silence transposons, but it is unclear how these pathways adapt to invasion of new transposons. In Drosophila, piRNAs are encoded by heterochromatic clusters and maternally deposited in the embryo; paternally inherited P elements escape silencing and trigger hybrid sterility syndrome: P-M hybrid dysgenesis. Novel piRNAs are associated with reduced transposition. The P elements trigger their own silencing.

Long interspersed nuclear element

Retrotransposons found in human genomes. With SINEs, comprise 40% of the genome. Mostly inert, and do not move in somatic cells, but are mobile in early embryos and in stem cells. Transposition is suppressed by heterochromatin and non-coding RNA.

Endogenous retrovirus (ERV)

Long terminal repeat retrotransposon

Found in most eukaryotic organisms. Resemble retroviruses. Move by synthesizing an RNA from the integrated DNA, which is then converted into dsDNA by the transposon-encoded reverse transcriptase. The DNA is then integrated at another landing site by homologous recombination. The LTR is also found in retroviruses. Studied in Schorn et al (2017). Responsible for the most TE insertions in mice. Expressed when histone H3K9Me3 is lost. More recent ERVs are less abundant in the genome.

Methyl-CpG binding protein 2

An X-linked gene. 70 - 80% of sporadic Rett syndrome cases are caused by mutations. In mecp2-/- mice, the induced expression of MeCP2 in late stages of development reversed symptoms of Rett syndrome. Studied in Gabel et al (2015). Represses transcription in a length-dependent manner. Has preferential binding for methylated CA sites, rather than CG sites. The CA sites must be methylated by Dnmt3a for MeCP2 to bind. MeCP2-induced genes have no common features, but MeCP-2 repressed genes are longer on average in the genome. In knockout mice, long genes over 100 kb are upregulated in brain regions compared to wildtype. In overexpressing mice, genes over 100 kb are downregulated compared to wildtype.

Germline mutation in NRP2 (NALP2) in a familial imprinting disorder (Beckwith-Wiedemann Syndrome)

Report on a family with Beckwith-Wiedemann syndrome, and an IC2 epimutation in which affected siblings had differential paternal 11p15.5 alleles excluding an in cis mechanism. Using a positional-candidate gene approach, found that the mother was homozygous for a frameshift mutation in exon 6 of NRP2.

P element

Cut-and-paste transposons found in Drosophila. P⁺ genotype is inherited through P⁺ males when they are mated to naive (P^-) females.  It is active only in zygotes from a cross of a P⁺ male and a P^- female. The progeny display dysgenesis, with multiple body disorders, and are often sterile. The transposon invaded the flies in the mid-20th century. Most wild flies are P⁺. P^- strains are found in labs which established their collections before 1950. P elements are activated only in cells with a naive cytotype. The P element is 2.9 kb with 31 bp inverted terminal repeats. Encodes a transposase gene interrupted by 3 introns. When mobilized in a naive zygote, it activates resident transposons, causing wide-spread insertions and removals of all kinds of P-like elements. Invasion triggers heritable changes in genome structure that appears to enhance transposon silencing. The genome treats the genetic crisis as DNA damage, and attempts to conduct repairs.  Mobility is suppressed by chromatin structure and non-coding RNA PiWi. Transposons are mobilized for a short period, and repressed by 21 days after fertilization; this coincides with expression of PiWis from sites of transposons.

Eutharian mammals

Mammals apart from egg-laying mammals and marsupials. Silencing of the X_i chromosome is very stable, and CpG islands are heavily methylated. The cytosine methyltransferase inhibitor, 5-aza-deoxy-cytidine, reactivates X_i in placental mammals. Which of the two X chromosomes is silenced and which is activated can differ between cells of a female mammal. Females are X mosaics; X_p is reactivated and X_m is inactivated randomly at a late embryonic stage.

Poly(A) retrotransposon

A subclass of retrotransposons which lack long terminal repeats (LTR). Has two open reading frames which are transcribed into RNA, which is reverse-transcribed into DNA. Have a sequence of A:T base pairs at one end which is derived from the poly(A) tail of retroposon RNA. Not related to viruses; descended from mRNA of ancient genes. Massively represented in mammalian genomes. The most abundant type of transposon in humans.

Copy-and-paste transposon

Transposable elements which move through an RNA intermediate. Have a common origin with retroviruses. Encodes an RNA which is reverse transcribed into DNA which acts as an invasive molecule. Has homologous recombination. Not found in prokaryotes. Studied in Schorn et al (2017). When active, known to cause genomic instability by reverse transcribing and inserting into the genome. Normally silenced by epigenetic marks. Includes LTR-retrotransposons and retroposons.

LTR-retrotransposon control by tRNA-derived small RNAs

During embryonic development, when stem cells undergo epigenetic reprogramming, control of transposable elements is lost; it is unclear how transposition is avoided. Determined the expression of sRNAs in early stem cells and how retrotransposon activity can be repressed by small tRNA-derived fragments (tRFs). Determined a mechanism of silencing when cells undergo genome-wide epigenetic reprogramming, and release transcriptional control of TEs. Findings can be applied to studies on cancer, and developmental and viral defence.

Short interspersed nuclear element

Retrotransposons found in human genomes. With LINEs, compromise 40% of the genome. Mostly inert, and do not move in somatic cells, but are mobile in early embryos and in stem cells. Transposition is suppressed by heterochromatin and non-coding RNA.

Transposon

A movable DNA element. Discovered by Barbara McClintock. Flanked by inverted repeats which are targeted by transposase. Found in the genomes of all organisms. In many species they constitute a large fraction of the total DNA. Account for 40% of the human genome. There are thousands in the genome. Different classes are described, with different mechanisms of mobility: includes cut-and-paste and copy-and-paste transposons. When it lands inside another gene it can cause problems. Known to harm the genome, yet they are tolerated, mostly by chromatin structure. Different species have different strategies to suppress transposons. Silenced by repressive heterochromatin through the RNAi pathway. Silencing is critical for maintaining genome stability. Can be active in early embryo cells, which lack heterohromatin; additional mechanisms act to restrict their mobility. At 4-cell stage, mobility diminishes and silencing becomes epigenetically stable. Tends to move to positions where it has been before, due to remnants of flanking sequences left at the site. Parasitic DNA; it moves for the sake of moving, and will move if allowed to. In bacteria they may confer antibiotic resistance. Evolve rapidly, and can mobilize and trigger genetic instability. Occasionally, can awaken or cross species, causing genetic disorders and diseases. Number of transposons increases with age, leading to genome instability. Studied in Wood et al (2016) and Elsasser et al (2015).

Chromatin-modifying genetic interventions suppress age-associated transposable element activation and extend lifespan in Drosophila

Studied the RNAi pathway that suppresses transposable elements. Dicer processes dsRNA into siRNA which is loaded onto an Ago containing a RISC complex, which catalyzes formation and spreading of heterochromatin with Sir2. Provides further evidence for the RNAi theory, and explores possible ways of controlling TE reactivation through dietary, genetic, and pharmacological methods. Importance of repression in heterochromatin is due to extensive distribution of TEs, which have the ability to accumulate over time if unchecked. Many heterochromatic genes and their associated TEs increase in expression in conjunction with aging, and with increases in body fat. Control of silenced heterochromatin and associated repression of TE activation allows for effective genomic stability, and contributes to longevity of genes, supported through dietary restriction and genetic interventions. Interventions reduced increases in TE activity related to age, and extended lifespan. Dietary restriction enhances lifespan by its effect on heterochromatin.

X-controlling element

An element of XIC. A cis-acting locus that biases random inactivation ratios. An enhancer. How it works is unknown.

X-inactivation specific transcript

An element of XIC. Encodes a 19 kb poly(A)+ untranslated RNA transcript. Expressed at high levels from X_i, but not from X_a. The RNA transcript coats X_i, but not X_a. Necessary for the initiation of X-chromosome inactivation in cis (expressed from the same chromosome that is inactivated). Not needed for maintenance of X-chromosome inactivation; transmission of chromatin state does not depend on Xist. In an undifferentiated cell, Xist is expressed from both chromosomes, but does not accumulate. As differentiation initiates, Xist is expressed from both, and accumulates around X_i. Once fully differentiated, it is expressed from X_i only, and coats X_i. Once large amounts of Xist wraps X_i, hypoacetylation and DNA methylation of the entire chromosome follows. Initiates a cascade of events that leads to complete inactivation. There is repression and late replication in somatic cells. It may form a molecular cage, or act as an attractor of silencing factors. Eventually the X_i chromosome shrinks into a Barr body.   

CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator of IGF2 imprinting in tumours

Studied IGF2 and ICR. Identified a novel interaction between the oncogenic microRNA, miR483, and promoter 2 (P2) in IGF2 promoter complex. Suggest that this interaction plays a significant role in altering the gene's epigenetic landscape, reactivating the maternally imprinted IGF2. May direct future research towards miRNA and potential roles in modulating epigenetic makeup of genes through imprinting.