1. SGN22 Regulation of Eukaryotic Genomes (CH 15.2, 15.3)

2. Regulation of gene expression in eukaryotes
Regulation of gene expression in eukaryotes
Genes must be regulated (turned on and off) for a number of reasons and in a number of ways
How are genes turned on and off selectively, especially in the case of multicellular organisms wherein each different cell type shares the same genes?
Two types of gene expression, each requiring control: Regulated and Differential Gene Expression

3. Genes must be turned on and off in response to extracellular stimuli and regular cycles (regulated gene expression)

4. Differential gene expression involves the transient activation of certain genes during development, and the turning on and turning off of certain genes indefinitely in the process of cellular differentiation (cell specialization), especially in multicellular organisms

5. Control of regulated gene expression
Control of regulated gene expression* occurs on many levels; in part made possible by decoupling of transcription and translation *Differential gene expression will be discussed more in SGN 23
Epigenetics - chromatin modifications
Chemical modification of DNA influences gene expression in regard to responses to stimuli and cellular cycles
Transcription initiation
Immediate and long term regulation of gene expression primarily takes place during transcription initiation
Post Transcriptional control
Control can occur at mRNA and protein level, including epigenetic mRNA silencing

6. Review eukaryotic gene and eukaryotic genome
Promoter binds polymerase and also transcription factors

7. Epigenetics at the level of chromatin modifications related to environmental signals and cell cycles affect the availability of genes for transcription
DNA methylation and Histone acetylation
Genomic imprinting/epigenetic inheritance
Imprinted genes are silenced

8. DNA methylation – attachment of methyl groups (-CH3) to DNA seems to inhibit transcription in most cases
Involved in long term inactivation of genes and chromosomes/chromosomal regions; seen in X chromosome inactivation
Methylation patterns pass on during cell division, meaning methylation patterns in the embryo are passed down from cell to cell as tissue differentiates

9. Histone acetylation – attachment of acetyl groups (-COCH3) to histones; causes histones to hold DNA less tightly, allowing transcription factor access

10. Heterochromatin (associated with methylation) is name for portions of chromatin that is tightly wound and inaccessible, while euchromatin is acetylated and less tightly wound, allowing access by transcription machinery

11. Genomic imprinting/epigenetic inheritance – inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence (hereditary patterns of histone acetylation, DNA methylation, etc.); can be important in diseases such as cancer or schizophrenia

12. Transcription initiation is a primary step of control of regulated gene expression
Transcription initiation is controlled by proteins that interact with DNA and with each other, called transcription factors
Transcription factors are typically proteins, although can be other biomolecules, such as steroid hormones

13. Typical eukaryotic gene
Consists, in part, of promoter, introns and exons; promoter upstream from coding region allows binding of RNA polymerase, which is part of transcription initiation complex
Also proximal and distal control elements also important in binding of TIC
Does not have operator so no simple repressor; more complex bimolecular interactions responsible for regulation

14. Transcription can typically only commence when transcription factors bind to RNA polymerase to form transcription initiation complex (compare to prokaryotic use of operator)

16. Still transcription is not efficient without role of control elements; some are proximal to promoters and some are distal (called enhancers); in many cases additional transcription factors (activators) bind to enhancers and cause DNA to bend so activators can interact with transcription initiation complex, thus stimulating transcription

17. Some trans factors act as repressors - can block control elements, bind activators or otherwise inhibit formation of TIC
Distal repressors
Proximal repressors

18. Trans factors often have similar structures – DNA-binding domain and protein-binding domains, allowing numerous proteins to join together in TIC

19. Regulated and differential gene expression both use transcription initiation as point of control Regulated gene expression involves turning genes on and off in response to extracellular stimuli and regular cycles Transcription initiation has “on/off” Differential gene expression involves the transient activation of certain genes during development, and the turning on and turning off of certain genes indefinitely in the process of cellular differentiation (cell specialization), especially in multicellular organisms In specialized cells inactivated genes permanently “off” Permanently “off” genes different in different cell types There is definitely overlap in regard to both of these types of expression Both involve response to extracellular signals RGE – all of the time DGE – during differentiation Cells are differentiated because of different regulated gene expression in different cell types

20. Regulation of transcription initiation allows for coordinated regulated control – coordinated activation of genes that produce proteins (most often enzymes) involved in same biochemical pathway
In eukaryotes genes that make proteins are involved in the same pathway are often scattered about the genome (for example genes involved in initiating the cell cycle); related genes will have control elements that respond to the same activators and transcription factors, so their transcription can be coordinated

21. Differentiated control - Differentiated cells must only turn on cell type specific genes (liver cell turn on liver genes, kidney cell turn on kidney genes, etc.); a differentiated cell will only have activators for cell type specific genes, so other genes will not turn on

22. Differentiated control involved in organismal development (zygote  blastula  gastrula  fetus, etc.) and cell maturation (for example, maturation of white and red blood cells)

23. Example – Testosterone (which enters cells and
Example – Testosterone (which enters cells and binds to intracellular receptors, forming an activator) permeates every cell of the male body but only certain cells will respond to it because only these cells have the receptors
In cells without the receptors the activators are not formed and the genes are not activated, but in cells with receptors multiple genes involved in the response to testosterone will be turned on

24. Post-transcriptional mechanisms play supporting roles in the control of gene expression; this involves everything between production of primary mRNA transcript and control of concentration and lifespan of protein product

25. Alternative RNA splicing by regulatory proteins specific to different cell types can produce many different proteins from one gene
Often alternate proteins are related; alternate mRNA splicing often retains many common protein domains (for example intramembrane domains) while mixing other domains (for example extracellular domains)

27. Protein synthesis can be affected by how long mRNA persists
mRNA degradation begins with molecular tagging of targeted mRNA, allowing for removal of poly-A tail and 5’ cap, followed by enzymatic digestion of mRNA; small noncoding RNAs (microRNAs –miRNA) are involved in tagging mRNA for degradation

28. Translation can be prevented when proteins and miRNAs bind to 5’ UTR or 5’ cap, and in some instances to 3’ poly A tail, stopping mRNA from joining with ribosomes; important in establishing orientation of embryo In some cases (ex eggs or plant cells in darkness) mRNA is stored in inactive form until needed; removal of bound inhibitory proteins or other steps allow translation initiation

29. Polypeptide processing and degradation
Regulation can occur at any of the steps involved in modifying (cleavage to make final polypeptide, addition of sugars or phosphate groups, etc.) or transporting a protein
Proteins can be selectively degraded (ex. cyclins), often by tagging them with a protein called ubiquitin, which signals protein to be destroyed by large proteasome protein complex (mutations can cause cancer)

30. Significant amount of transcribed RNA (together with tRNA and rRNA called noncoding RNA - ncRNA) is transcribed but not used to make proteins; instead used in epigenetic regulation and other ways
Several categories of small RNA’s that inhibit translation or promote mRNA degradation are being discovered (miRNA, siRNA, piRNA, etc.)

31. MicroRNAs (miRNA) and small interfering RNAs (siRNA) – short RNA sequence that forms a complex with protein, allowing complex to bind to complementary mRNA strand, keeping it from being translated and/or targeting it for destruction
siRNA and other types of ncRNA are also thought to be involved in heterochromatin formation, including genomic imprinting