gene regulation

see also:

• micro RNAs (miRNAs)
• DNA and genes

• for a cell to function properly, necessary proteins must be synthesized at the proper time.
• gene regulation is the process used to control the timing, location and amount in which genes are expressed as RNA and as a result protein is produced
• only 1% of the genome are genes that are used to manufacture proteins, most of the remainder are utilised for the regulation of gene expression and protein manufacture
• genetic regulation in bacteria:
  • promoter gene
  • operator gene
  • interplay of feedback loops acting upon gene expression repressors and activators

• unlike bacteria, eukaryotic genes are not organized into operons, so each gene must be regulated independently at various levels of regulation:
  • epigenetic changes
    • alter the chromosomal structure so that genes can be turned on or off
    • eg. epigenetic alteration of histone protein gene can affect DNA transcription as histone can expose or hide DNA segments
      • eg. Setd1b - a gene responsible for producing a protein that adds methyl groups to histone H3 at lysine 4, resulting in the H3K4me3 epigenetic modification, and upregulation of Myc genes, which plays a critical role in regulating cell growth, division, and metabolism and can contribute to cancer development if altered, especially AML ¹⁾
    • eg. TET1 (an enzyme responsible for DNA demethylation) is a pivotal player in epigenetic regulation, in the development and progression of various clinical diseases²⁾
      • vitamin C helps maintain TET activity by donating electrons to regenerate Fe²⁺ from Fe^{3+ 3)}
    • eg. LINE-1 retrotransposon elements
    • eg. removing the enzymes responsible for adding two epigenetic markers called H3K79 methylation and H3K36 trimethylation resulted in over-expression of YAP-TEAD proteins and blocked the stem cell's ability to become neurons⁴⁾
  • transcriptional
    • histone alterations and variations in DNA segment exposure and folding
      • histone post-translational modifications
        • histone acetyltransferases (HATs) catalyse histone lysine acylation which is involved in essential biological activities, such as transcriptional regulation, DNA-damage repair, and cell-cycle progression
          • abnormal acylation is strongly associated with various diseases, such as cancer
    • RNA polymerase requires other proteins, or transcription factors, to facilitate transcription initiation
    • transcription factors are proteins that bind to the TATA box section within a gene's promoter sequence and other regulatory sequences to control the transcription of the target gene
    • transcription factors must bind to the promoter region first and recruit RNA polymerase to the site for transcription to begin - RNA polymerase cannot do this alone
    • the promoter region is immediately upstream of the coding sequence and contains the TATA box. This box is simply a repeat of thymine and adenine dinucleotides (literally, TATA repeats)
    • in some eukaryotic genes, there are regions that help increase transcription. These regions, called enhancers, are not necessarily close to the genes; they can be located thousands of nucleotides away in which case they are brought into proximity by folding of the DNA via histone protein
    • transcriptional repressors can bind to promoter or enhancer regions and block transcription
    • examples:
      • intracellular glucocorticoid receptor (GR)⁵⁾
        • ligand-activated glucocorticoid receptor (GR) induces or represses the transcription of thousands of genes through direct binding to DNA response elements, physically associating with other transcription factors, or both.
        • there are many GR variants which are derived from a single gene by alternative splicing and alternative translation initiation mechanisms
        • posttranslational modifications of these GR isoforms further expand the heterogeneity of glucocorticoid signaling
        • the central DBD of the GR is the most conserved domain across all the nuclear receptor proteins and harbors two zinc finger motifs that bind target DNA sequences called glucocorticoid response elements (GREs) which are palindromic sequences comprised of 2 half sites (GGAACAnnnTGTTCT) separated by a 3-nucleotide spacer. GR binds GRE as a dimer and each half site is occupied by one receptor and thus the 3-nucleotide spacer between the 2 half sites is strictly required for GR:DNA interaction.
        • there is also a negative glucocorticoid-responsive element (nGRE) that mediates glucocorticoid-dependent repression of target genes by recruiting co-repressors (NCoR1 and SMRT) and histone deacetlyases (HDACs). nGRE is palindromic (CTCC(n)_0-2GGAGA), but differ from the classic GRE in having a variable spacer that ranges from 0-2 nucleotides and is occupied by 2 GR monomers
        • the NTD part of GR contains transcription activation function (AF1) that activates target genes in a ligand-independent fashion and is the primary site for all the posttranslational modifications
        • in the absence of hormone, GR predominantly resides in the cytoplasm of cells as part of a large multi-protein complex that includes chaperone proteins (hsp90, hsp70, and p23) and immunophilins (FKBP51 and FKBP52)
        • on binding ligand GR undergoes a conformational change, resulting in the dissociation of the multi-protein complex leading to a structural reorganization of the GR protein exposing the 2 nuclear localization signals, and the ligand bound GR is rapidly translocated into the nucleus through nuclear pores. Binding of glucocorticoids to GR not only activates the receptor, but also liberates accessory proteins that participate in secondary signaling cascades (eg. c-Src activates signaling cascades that inhibit phospholipase A2 activity, phosphorylate annexin 1, and impair the release of arachidonic acid)
        • Once inside the nucleus, GR binds directly to GREs and stimulates target gene expression.
        • specific GR binding sites vary between tissues due to differences in chromatin landscape which influences GRE accessibility
        • GR transcriptional activity can be regulated in many ways:
          • inheritable polymorphisms in the GR gene that alter the amino acid sequence are linked to impaired GR function as a transcriptional activator or repressor.
            • N363S polymorphism, located within exon 2 occurs in ~4% of the population, results in modest increases in GR transcriptional activity, and is associated with generalized increases in glucocorticoid sensitivity. Carriers have been reported to have an increased body mass index, coronary artery disease and decreased bone mineral density.
            • ER22/23EK polymorphism that occurs in ~3% of individuals results in an arginine (R) to lysine (K) change at position 23 (R23K) within the N terminus and is associated with decreased GR transcriptional activity and has been shown to increase the ratio of GRα-A to GRα-B and the carriers of ER22/23EK polymorphism have a lower tendency to develop impaired glucose tolerance, type-2 diabetes and cardiovascular disease
            • A3669G polymorphism in GRβ 3’ untranslated region results in an increase of both GRβ mRNA and protein. Carriers of A3669G polymorphism have a higher incidence of rheumatoid arthritis and cardiovascular disease. Those homozygous for A3669G polymorphism were associated with a pro-inflammatory phenotype with an increased risk for myocardial infarction and coronary heart disease
          • alternative splicing GR variants (GRα, GRβ, GRγ, GR-A, and GR-P)
          • translational GR isoforms (GRα-A, -B, -C1, -C2, -C3, -D1, -D2 and -D3)
          • down-regulation of GR
          • covalent phosphorylation of GR modifies other properties of GR that affect GR signaling
          • GR is ubiquitinated at a conserved lysine residue located at position 419 (Lys-419), and this modification targets the receptor for degradation by the 26S proteasome
            • mutation of this conserved Lys residue enhances the glucocorticoid-induced transcriptional activity of GR and blocks ligand-dependent down-regulation of GR
          • acetylation of GR by clock transcription factor reduces GR transcriptional activity
          • covalent addition of a small ubiquitin-related modifier-1 “sumoylation” which can promote its degradation and inhibit the transcriptional activity of GR in a promoter-specific manner by recruiting co-repressors
        • in thymocytes, activated GR translocates to mitochondria and regulates apoptosis
        • additionally, GR can physically interact with the members of the signal transducer and activator of transcription (STAT) family, either in conjunction with binding a GRE or apart, to enhance transcription of certain target genes
  • nuclear shuttling
  • post-transcriptional
    • occurs after the mRNA is transcribed but before translation begins
    • alternative RNA splicing occurs with 70% of human genes and is a mechanism that allows different combinations of introns, and sometimes exons, to be removed from the primary transcript allowing different protein products to be produced from one gene
    • control of the half life of mRNA - mRNA stability contributes to amount of protein produced
      • RNA-binding proteins (RBPs)
        • can bind to the regions of the RNA just upstream or downstream of the protein-coding region.
        • These regions in the RNA that are not translated into protein are called the untranslated regions, or UTRs.
        • The region just before the protein-coding region is called the 5′ UTR, whereas the region after the coding region is called the 3′ UTR (Figure 17.12).
        • The binding of RBPs to these regions can increase or decrease the stability of an RNA molecule, depending on the specific RBP that binds.
      • micro RNAs (miRNAs)
        • can also bind to the RNA molecule along with a ribonucleoprotein complex called the RNA-induced silencing complex (RISC) which rapidly degrades the target mRNA
  • translational
    • mRNA binding to the ribosome
    • shuttling of mRNA-ribosome complex to endoplasmic reticulum (ER)
      • the first few amino acids proteins destined for ER are a tag called a signal sequence.
      • as soon as these amino acids are translated, a signal recognition particle (SRP) binds to the signal sequence and stops translation while the mRNA-ribosome complex is shuttled to the ER where the SRP is removed and translation resumes
  • post-translational
    • modifying the protein after it is made
      • enzyme inhibition mechanisms
      • activity and/or stability of proteins can also be regulated by adding functional groups, such as methyl, phosphate, or acetyl groups
      • addition of an ubiquitin group to a protein marks that protein for degradation and then are moved to a proteasome, an organelle that degrades proteins

• induced pluripotent stem cell (iPSC) technology
  • invented in 2006, this technique uses stem cells to create target cells
  • takes ~ 1month and there is a risk of tumour formation and immune rejection
• direct re-programming of cells
  • invented in 2025 by a Japanese team⁶⁾ who re-programmed the expression of 4 genes, Nkx2-1, Foxa1, Foxa2, and Gata6 into mouse embryonic fibroblasts, and created induced pulmonary epithelial-like cells (iPULs) which are similar to lung alveolar epithelial type 2 (AT2) cells which successfully transplanted into mouse lungs with interstitial pneumonia, some of the cells had differentiated into alveolar epithelial type 1 (AT1)-like cells, which are essential for lung tissue regeneration
  • this technique could be used for autologous transplants with less immune rejection and less tumour formation risk than iPSC