The Non Coding Genome

What is the relevance of transcriptional control?

• controlling many fundamental biological processes by ensuring the correct expression of specific genes

• cells become different during development because of the regulated expression of thousands of different genes in time and space causing stem cells to adopt different fates and differentiate into more specialised cell types

• diseases areas from a breakdown in the transcription regulatory system

What genes does RNA polymerase I transcribe?

5.8S, 18S, and 28s

What genes does RNA polymerase II transcribe?

• all protein coding genes

• snoRNA genes

• miRNA genes

• siRNA genes

• lncRNA genes

• most snRNA genes

What genes does RNA polymerase III transcribe?

• tRNA genes

• 5S rRNA genes

• some snRNA genes

• genes for other small RNAs

What is the process of RNA pol II transcribing genes

• RNA pol II is a large 12 subunit protein complex

• recruited to target genes along with General transcription factors and regulatory proteins in a complex called RNA pol II holoenzyme (INITIATION)

• RNA pol II moves stepwise along the DNA unwinding the double helix at its active site

• complementary nucleotides are added in a sequential manner using the anti sense DNA strand as a template (ELONGATION)

• RNA pol II stops at the end of a gene and is released from the DNA strand (TERMINATION)

Gene promoters

• genomic DNA sequences that define the position where transcription of a gene by RNA pol II begins: transcription start site (TSS)

• typically located directly upstream of the target gene

• contain a number of DNA sequence motifs that are bound by the GTFs and RNA pol II in a stepwise manner

• GTFs position RNA pol II at the correct location on the genome to initiate transcription

• different classes of promoters exist and a given promoter does not necessarily contain all of these motifs

Role of enhancers and silencers in transcription accuracy?

• DNA sequences located on the same chromosome as the genes they regulate

• can be located upstream or downstream of their target genes and within introns and coding regions

• can be located many kilobases away from their target promoters (distal)

• modulate the rate of promoter transcription

How do distal cis-regulatory sequences communicate with their targets promoter?

• Transcription factors proteins read the sequence of cis regulatory DNA and bind to specific motifs

• DNA bound TFs interact with GTFs and RNA pol II assembled at the promoter

• involve contacts with intermediary proteins called transcriptional coactivators and corepressors that don’t directly bind DNA

• intervening DNA looped out

• enhancer elements speed up rate of complex assembly and silencers block rate of complex assembly

Insulator Elements

• class of DNA sequences that function to prevent the innappropiate regulation of adjacent genes

• insulator situated between an enhancer and a promoter

• block the action of a distal enhancer on a promoter

• control the gene or set of genes that an enhancer can regulate

Barrier elements

specialised type of insulator sequences that prevent the spread of condensed chromatin sequences

How are correct spatial and temporal gene expression patterns created?

• e.g Msx1 homeodomain transcription factor is an important regulator of pluripotency

• expressed in mesenchymal progenitor cells in the developing embryo (differentiate into a variety of cell types, including: osteoblasts, chondrocytes, myocytes, and adipocytes)

Transcriptional Control in the Post Genomic Era

• final sequencing map of the human genome released in 2003 defining the start of post genomic era

• multiple large scale international collaborations established to identify and annotate all the functional DNA elements in the non coding genome

ENCODE Project

• multiple experimental techniques, genome sequencing and computational analysis to map regulatory elements genome wide

  • 15.2% of the genome contains open chromatin

  • 8.1% is bound by a transcription factor

  • 399,124 putative cis regulatory DNA sequences

  • 70,292 regions with promoter like feature

  • genome expresses many thousands of non-coding RNAs in addition to protein coding genes

Classes of non-coding RNAs that genomes encode

• micro RNAs

• siRNAs

• lncRNAs

• Piwi interacting RNAs

Long Non Coding RNAs

• greater than 200nt in length

• do not encode for a protein

• possess a polyA tail and 5’7methylguanosine cap

• fewer exons and shorter transcript length compared to mRNAs

• more tissue restricted expression than mRNAs

• located in either the nucleus or the cytoplasm

FANTOM

• Identified 27919 high confidence human lncRNA genes

• most transcriptional enhancers generate a lncRNA

• many intergenic lncRNAs are coexpressed with nearby protein coding genes and overlap expression quantitative trait loci suggesting that they may act in cis to regulate the transcription of nearby genes

• a subset of lncRNAs have emerged as a new class of gene expression regulators