Transcription Regulation Notes

Why is Transcriptional Regulation Needed?

• Allows for the development of different tissues.
• Facilitates the transition from childhood to adulthood.
• Enables reaction to environmental cues.
• Deregulation can lead to uncontrolled growth, such as in cancers.
• Example: transition from fetal to adult hemoglobins, involving changes in expressed protein subunits and coordinated functions post-birth.

What Determines When/How Genes Are Transcribed?

• Continued chromatin structure.
• RNA polymerase and general transcription factor binding specificity.
• Additional binding and activation factors.

Heterochromatin

• Expressed genes are found in an ‘open’ conformation (euchromatin).
• Genes within highly packed heterochromatin are usually not expressed.
• Histone code involves silencing and activation.
• Specific constitutive heterochromatin structures: origins of replication, telomeres, and centromeres.
• Facultative heterochromatin - cell-type-specific, can switch into euchromatin following developmental cues.

Heterochromatin and the Histone Code

• $H3K27me3$: Inactivation of HOX genes and X chromosome inactivation.
• $H3K9me$: Associated with organogenesis.
• $H3K27me3$ binds ‘polycomb’ proteins, which remodel chromatin.
• $H3K4me2$: Centromeric.
• Constitutive heterochromatin: regions consistently silenced in all cell types, such as centromeres, telomeres, some transposons, and gene-poor regions; governed by histone methyltransferases.

Coding Back to Euchromatin

• $H3K4me$ and $H3K9ac$: Gene expression.
• $H3S10p$ and $H3K14ac$: Gene expression.
• Actively expressed regions (green) are central in the eukaryotic nucleus.
• Heterochromatic regions (red) are close to the nuclear membrane and associated with lamins.
• DNA can be moved to the nuclear lamina/membrane for transcriptional inactivation.

Three Functional Heterochromatic Elements

• Telomeres
• Replication origins
• Centromeres

Human Centromere Organization

• Normal $H3K4me2$
• Centromere-specific H3
• Pericentric and centric regions
• Microtubules and cohesin linking sister chromatids
• Kinetochore
• Centric heterochromatin: long, highly repetitive chromatin structures.
• $H3K4me2$ allows an open structure, permitting kinetochore attachment.

Telomeres

• Telomeres are repetitive structures that vary in length and DNA sequence.
• Examples of telomere repeats:
  • GGGTTG in the ciliate Tetrahymena
  • GGGTTA in vertebrates
  • $G_{1-3}A$ in Saccharomyces cerevisiae.
• Vertebrate sequences are repeated over several kb; yeast telomeres are several hundred bp.
• Telomeres shorten after each cell division due to the 5’ to 3’ synthesis of DNA and the erasure of RNA primers.

Telomerase

• Telomerase binds the single-stranded G-overhang, and is a ribonucleoprotein (RNP) enzyme.
• Composed of telomerase RNA (TER) and telomerase reverse transcriptase protein (TERT).
• Telomerase extends the 3’ end of the parental strand using its own RNA subunit as a template.
• Tom Cech discovered TERT.

Compensatory Mechanism for Telomere Shortening

• RNA-templated DNA synthesis.
• DNA primase lays down an RNA primer on the extended G-overhang.
• DNA-templated DNA synthesis by DNA Polymerase extends this primer 5’-3’.
• DNA ligase ligates the new Okazaki fragment to the old lagging strand 5’ end.
• There is still a free 3’ unpaired end, which normally triggers repair mechanisms.
• Extended G-overhang.

Shelterin Complex / Telosome

• The 3’ end shelters from repair mechanisms by base pairing.
• A shelterin complex includes TRF1 (telomeric repeat-binding factor 1), TRF2 (telomeric repeat-binding factor 2), RAP1 (repressor/activator protein), and others.
• Stimulates t-loop formation.
• Displaces a d-loop and results in the base pairing of the 3’ end.
• The 3’ end shelters from repair mechanisms in a telosome.

Werner Syndrome

• Werner syndrome occurs if we cannot replicate our telomeres.
• Inheritance: autosomal recessive
• Incidence: 1 in 1,000,000, but in Japan and Sicily, it is 1 in 30,000.
• The WRN helicase protein is important for telomeric DNA replication.
• Telomeres replicated by lagging strand synthesis are not efficiently replicated in Werner cells.
• Overexpression of telomerase maintains telomere length in Wrn cells.
• The WRN helicase protein is also important for DNA repair, resolving recombination structures (Holliday junctions), and governing Okazaki fragment joining in eukaryotes.

How is Transcription Controlled?

• What determines how/when/why genes are transcribed?
• Chromatin structure
• RNA polymerase (and general TF) binding specificity
• Additional binding and activation factors

Eukaryotic RNA Polymerases

• Relatively scarce, about 0.001% of total cell protein.
• Highly complex, typically 12 subunits (compare with bacterial: five subunits).
• Three different enzymes, functionally and biochemically distinct:
  • RNA polymerase I (Pol I): 5.8S, 18S, 28S rRNA genes
  • RNA polymerase II (Pol II, RNAP): all protein-coding genes, snoRNA genes (small nucleolar), miRNA genes (micro), siRNA genes (small interfering), most snRNA genes (small nuclear)
  • RNA polymerase III (Pol III): tRNA genes, 5S rRNA genes, some snRNA genes, other small RNA genes
• RNA polymerases in bacteria, archaea, and eukaryotes are closely related: the basic features of the enzyme were in place before the divergence of the three major branches of life.
• Structural similarity: bacterial RNAP and eukaryotic RNA Pol II.
• Grey: extra eukaryotic subunits.

Pol II Promoter

• RNA Pol II promoter regions contain multiple cis-acting elements that bind proteins; individual elements may not always be present; Pol II promoters are very variable.
• 'core' promoter regions, ~ -50 to +50 bp are depleted of nucleosomes: accessible.
• Elements include:
  • BREd (downstream B recognition element): binds TFIIB
  • TATA box: binds TATA-binding protein (TBP)
  • BREu (upstream B recognition element): binds TFIIB
  • Inr (initiator element): binds TFIID

TBP (part of TFIID)

• TBP binds the TATA box, and the strength of binding regulates transcription.
• BREd binds TFIIB
• TATA box binds TBP
• BREu binds TFIIB
• Inr binds TFIID

TATA Box

• The TATA box is a consensus sequence: individual TATA boxes have different affinities for TBP – and so some are more efficient at stimulating transcription than others.
• The TATA box is a consensus sequence.
• Individual TATA boxes have different affinities for TBP, so some are more efficient at stimulating transcription than others.

G-less Cassette Transcription Assay

• A radioactive RNA transcript of a defined size (typically ~ 400 bp) is produced.
• It can be electrophoresed through polyacrylamide gels and quantified following autoradiography.
• Principle: Because no GTP is supplied, the RNA is truncated at the point at which a ‘G’ should be inserted.

Different TATA Sequences Support Different Levels of Transcription

• TATAAAA (AdML): Human TFIID binds strongly and supports high expression of the G-less cassette.
• TATAAAG (Yeast His): Human TFIID binds less strongly to the yeast His TATA box - reduced expression.

TATA Boxes Regulate Expression

• Even in viruses.
• EBV productive cycle:
  • Immediate Early (IE) and Early (E) genes (n = 35)
  • Late (L) genes (n = 33)
• L genes have a distinct TATT motif, but IE and E genes have a TATA motif, providing temporal control.
• IE and E genes are differentially expressed, indicating other control sequences.

EBV and cis-acting elements

• Typical L gene: TATTAA, minimal complexity
• Typical E gene: TATAAA, intermediate complexity, TATA box with both proximal and distal positive cis-acting elements (green) that enhance transcription
• Typical IE gene: TATAAA, high complexity, TATA box with proximal positive cis-acting elements (green) that enhance transcription and both proximal and distal negative cis-acting elements (red) that inhibit transcription

Additional Binding and Activation Factors

• Activator proteins:
  • NF-κB
  • Interferon response factor
  • ATF-2/c-Jun

How is Transcription Controlled? (recap)

• What determines how/when/why genes are transcribed?
• Chromatin structure
• RNA polymerase binding specificity
• Additional binding factors
  • Activator/repressor proteins
  • Mediator proteins
  • Chromatin-modifying proteins

Additional Binding Factors – Activators (and Repressors)

• Eukaryotic RNA polymerases cannot access DNA directly: they require additional factors.
• General transcription factors assemble at the promoter and form a complex with RNA PolII.
• Specific transcription factors regulate the rate of transcription: activators increase transcription, repressors decrease transcription.
• These transcription factors bind the proximal promoter elements and the distal (enhancer) elements.
• They have a modular design: a DNA-binding domain that binds specific DNA sequences and an activating/repressing domain (protein interaction domain) that stimulates/inhibits transcription by interacting with mediator proteins, general transcription factors, or RNA PolII.
• DNA–binding domains:
  • Homeodomains
  • Zinc finger motifs
  • Leucine zippers

DNA-binding domains: homeodomains

• Helix 3 binds in the major groove of DNA making specific interactions between amino acids and nucleotides.

DNA-binding domains: Zinc Finger Motifs

• Zinc finger: a small structural motif with key Cys and His residues that coordinates a zinc ion ($Zn^{2+}$), stabilizing the fold.
• Often there is a cluster, arranged one after the other so that the α-helix of each binds the major groove of the DNA.
• A strong and specific DNA-protein interaction is built up through a repeating basic structural unit.

DNA-binding domains: leucine zippers

• Leucine zippers can also bind to DNA as heterodimers, expanding the potential regulatory repertoire.
• Three distinct DNA-binding specificities can be generated from two types of leucine zipper monomer, while six can be created from three types of monomer, and so on.
• Heterodimeric binding is common (e.g., Jun/Fos heterodimers).

Multimerisation and Combinatorial Control

• In T-cells (example):
  • AP-1 complex (c-Jun/c-Fos)
  • Nuclear factor of activated T-cells (NFAT)
• Combinatorial regulation is a powerful mechanism that enables tight control of gene expression, via integration of multiple signaling pathways that induce different transcription factors required for enhanceosome assembly.

What is the Enhanceosome?

• For genes that require tight control, activators bind cooperatively along an enhancer sequence, forming an enhanceosome.
• Each enhanceosome is unique to its specific enhancer.
• It recruits coactivators and general transcription factors to the promoter region of the target gene to begin transcription.
• It also recruits non-histone architectural transcription factors called high-mobility group (HMG) proteins, which regulate chromatin structure – they ensure that the target gene can be accessed by transcription factors.

Repressors

• In this example, four cis-acting elements, with four specific binding proteins, but their orientation and ability to bind co-activators and co-repressors increases the repertoire of responses

Putting it all together

• Expression of an RNA PolII-transcribed gene depends on the integrated output of:
  • DNA methylation status
  • Chromatin structure and histone modification status
  • General transcription factors and RNA Pol II
  • Regulatory complexes bound both upstream and downstream of the gene
  • Mediator proteins

Philadelphia (Ph) Chromosome

• All cases of chronic myeloid leukemia (CML) carry a Philadelphia re-arrangement.
• Inappropriate expression of a fusion protein that is permanently locked ON.

Burkitt’s Lymphoma

• Inappropriate regulation of an oncogene by a very strong enhancer.