Epigenetics - Appunti di lezione

LONG NON-CODING RNAs

Origin. They can be originated from intronic, exonic, intergenic, intragenic, promoter regions, 3’UTR, 5’UTRand enhancer sequences, the portion of the genome that encode them is not a protein encoding portion.they are anyway genes encoding for lncRNAs. They can be present in whatever position with respect ofprotein coding genes.

Example: Gene 1 and gene 2 are protein coding genes. There is also an intergenic region that contains an enhancer that regulates gene expression. lncRNA encoding region canbe located in whatever position: lncRNA can have the same orientationof the encoded gene (A arrow), can coincide with only a part of exon of gene 1 (B arrow), can include some exons and some introns of gene 1 (D arrow), the UTR region (E), can be oriented in the opposite direction(J), can be intergenic (F) or correspond to enhancer or promoter (G,H,I).

Characteristics and differences / analogies with protein coding genes:

• The region that encodes for lncRNA has the...

same organization of protein coding genes: promoter, exons and introns.

lncRNA have little evolutionary conservation with respect to protein-coding genes: the function is not strictly dependent on the sequence, even if can be important.

Mostly lncRNA are localised in the nucleus, but sometimes they can be exported to the cytoplasm.

Even small number of copies of lncRNA have an effect in regulatory role.

They are transcribed by RNA polymerase II as for the protein coding genes. They are regulated by some of the same transcription factors which regulate protein coding genes. The regulation of transcription can occur at epigenetic levels: the structure of the chromatin can be determined by histone modifications and chromatin structure and DNA methylation are important as in protein coding gene.

After the primary transcript is obtained, the majority of lncRNAs are spliced, polyadenylated and 5′-capped.

The expression levels are often low.

The transcription of lncRNA can regulate

The expression of protein-coding genes, in cis (in close genomic proximity) or in trans (to target distant loci). Gene expression regulation by lncRNA can occur through:

1. Recruitment of epigenetic regulators, leading to histone modifications, DNA methylation, chromatin remodelling => RNA sequences have structural flexibility, they can bind one or different proteins forming complexes that function as a unit. They can also bind to DNA. Many of the proteins involved in epigenetic regulation are not specific for DNA sequence, they require the presence of lncRNA to be associated to a specific DNA region.
2. Many LncRNA (> 20% in humans) bind both to DNA specific site and to a polycomb repressive complex or chromatin remodelling complexes that perform epigenetic modifications in these regions. lncRNAs bind specifically to DNAs: this occurs by 3 mechanisms:
1. Direct RNA-DNA interaction→ interact with the double strand of DNA forming triple strand structure.
2. lnc RNA -nascent transcript RNA

1. interaction through base pairing
2. indirect interaction lncRNA-DNA binding protein.

Some examples of known nlc RNA functions:

• Xist noncoding RNA that is involved in X chromosome inactivation can recruit PRC2.
• 2 imprinted loci examples:
• Air in mouse Igf2r imprinted gene cluster recruits H3K9 histone methyltransferase G9ato a specific promoter, then some genes are repressed.
• lncRNA that is expressed in another region that is imprinted in human and it controls the expression of neighbouring genes by recruiting H3K9 histone methyltransferase and PRC2 complexes.
• nlcRNA called HOTAIR => antisense transcribed in the HOXC locus, capable to regulate distally in trans another locus; the 5′ domain of HOTAIR physically interacts with PRC2 facilitating histone H3 lysine-27 trimethylation on the HOXD locus that is inactivated by this modification.

The same lnc RNA is capable to recruit at 3’ domain the demethylase LSD1 responsible for H3K4 demethylation associated with chromatin.

Condensation and gene inactivation. It induces 2 modifications, both repressive.

- ANRIL The INK4b-ARF-INK4a locus encodes two members of the INK4 family of cyclin-dependent kinase inhibitors, p15(INK4b) and p16(INK4a), and a completely unrelated protein, known as ARF. All three products participate in major tumour suppressor networks. ANRIL is an antisense non-coding RNA transcribed from the INK4b-ARF-INK4a locus. Polyadenylated RNA transcripts, spans 126.3 kb. The function is to bind to this locus and regulate p15 and p16 genes recruiting PRC2 and 1. PRC 2 perform the methylation of H3K27, PRC1 performs histone ubiquitination: it represses the tumor suppressors and facilitates oncogenesis.

2. Long noncoding RNA directly interfere with the binding of transcription activators/repressors to promoter. Ex of lncRNA DHFR => DHFR gene contains minor and major promoters. The major promoter is suppressed in quiescent cells. A minor promoter controls the transcription of lnc RNA that it is

Capable to bind both the major promoter and a transcription factor, TFIID, blocking the initiation of transcription of protein coding gene (it prevents the formation of the preinitiation complex. It can bind to DNA thanks to the formation of triple helix (double DNA and the strand of RNA). It is determined by sequence binding rules similar to the pairing rules of DNA strands. Triplex structure may be a common LncRNA mechanism for inhibition of promoter activity.

Enhancer-like lncRNAs (eRNAs) => some lncRNA are transcribed by RNA polymerase II from enhancer region and have functional properties of enhancer, activating gene expression. The levels of these unspliced transcripts tend to correlate positively with expression levels of neighbouring protein coding genes. So, the more the enhancer transcribes, the more it is capable to activate the transcription. eRNA can recruit transcription factor and transport the transcription factors to the promoters.

3. Regulation of alternative splicing:

Example of Zeb2 NAT => this lncRNA is complementary to 5’ splicesite of an intron of the gene, it can bind the primary transcript and mask the splicing site that is not recognised by splicing factors and it can affect the expression of the gene because the efficiency of translation by the ribosome can be different.

4. Effects on mRNA stability/degradation: Example concerning pseudogenes: PTENP1 and KRAS P.

Pseudogene = a sequence which is found in the genome that has the same sequence of a proper coding genome but without introns: it derives from a reverse transcriptase copy of a mRNA, is a cDNA inserted in the genome in a new position. Pseudogenes are not functional and not transcribed; they can have the function when they are transcribed by regulating the stability of mRNA of their paralogue genes (in this case PTEN, a tumor suppressor gene, and KRAS, a protooncogene:

• PTEN gene is transcribed and then mRNA is translated into PTEN protein. The process of translation is regulated.

iscapable to restore the original length. Telomerase uses an RNA template to make CDNA and to insert this in→the telomeres. Telomerase is a retro-transcriptase enzyme and it is regulated by lncRNA called TERRAlncRNA transcribed in the telomeric region. When the telomeres are short, the lncRNA is produced in smallamount because it is transcribed by telomeric region: when they are short, few copies of TERRA areproduced, in this case telomerase is active and can elongate the telomerase, when they reach a full length,more TERRA is transcribed and lcnRNA can interact with the telomerase enzyme reducing its activity. This isable to maintain the length in telomere in the correct range in the cells where the telomerase is active. 26NATs => natural antisense transcripts, RNA transcripts complementary toother RNA transcripts: if a certain protein encoding gene is transcribed inone direction, NATs is transcribed in the other direction. Transcriptional→interference if there is

antisense transcript, RNA polymerase that perform the transcription in both directions can interfere, there is a block in the transcription if there is an important antisense transcription: the expression of gene is affected because an antisense transcription is activated in the opposite direction. This mechanism includes RNA→interference there is a sense RNA and an antisense RNA with the formation of double strand RNA that is processed by dicers forming short interference RNA that can trigger the silencing of the gene. RNAs can target the gene region inducing silencing of it by inducing epigenetic modifications. These 2 last mechanisms are important.

LEZIONE 7

TRASPOSABLE ELEMENTS (TE)

TE => mobile genetic elements, DNA sequences able to insert in new genomic positions. They are present in all living organisms; in general, in complex species a higher percentage of the genome is represented by TE respect to others. In humans, they represent 45% of genome. TE can confer plasticity to the genome.

Transposons can have effects on genetic variability, on expression and epigenetic status of protein coding genes.

There are 2 main kinds of transposons that are classified as:

1. DNA transposons => class II, they insert themselves by a cut and paste mechanism.
2. RNA transposons => class I, they insert thanks to a copy and paste mechanism involving the role of a reverse transcriptase enzyme (retro transposition): RNA is reversely transcribed to cDNA and inserted in the genome in a new position, the old copy is maintained in the genome. At the end, you have an increased number of RNA transposon during evolution because the mechanism is based on adding new copies without removing the oldest. RNA transposons in vertebrates and mammals are much more important because they are partly active, while DNA transposons are present in ancient less copies not active today.

Retro-transposition is the mechanism that RNA transposons use to insert new copies of themselves in the genome. There is a role for retrotranscriptase enzyme.