VL 8: Polycomb Group silencing Flashcards
Polycomb Group proteins
Discovery:
* Polycomb (Pc) mutants were found to affect the expression of Hox genes.
* The Polycomb (Pc) protein was identified by Lewis in 1978.
Effects of Polycomb Mutants:
* In mutants, extra sex combs appear on the second and third legs.
* Homozygous mutants (having two copies of the mutant gene) are embryonic lethal. These mutants show transformation of all body segments toward the most posterior abdominal segment.
Role of PcG Proteins:
* PcG proteins are crucial for maintaining the repression of Hox genes, rather than establishing Hox gene activity.
* The initial establishment of Hox gene activity is done by transcription factors from early segmentation genes.
Consequences of Losing PcG Gene Activity:
* When PcG gene activity is lost, homeotic genes are expressed abnormally during early embryogenesis. This abnormal expression controls the identity of body segments.
For example, mutations can transform a segment into a different one, as seen in the antennapedia and bithorax complexes
PcG and Hox genes
Hox Genes:
* Determine the identity of body segments along the anterior-posterior axis in animals.
* Correct spatial and temporal expression is crucial for proper development.
Role of PcG Proteins:
* Maintain repression of genes that should not be expressed in specific cells or at certain stages.
* Modify chromatin to make it less accessible for transcription.
* Ensure proper segmental identity by repressing Hox genes where they should not be active.
* Maintain this repression through cell divisions (cellular memory).
Consequences of PcG Mutations:
* Lead to ectopic (abnormal) expression of Hox genes during early embryogenesis.
* Cause homeotic transformations (e.g., segment identity changes).
Examples:
Drosophila: Extra sex combs and segment transformations.
Mammals: Vertebral transformations.
Mechanism:
* PcG proteins maintain Hox gene repression but do not establish initial Hox activity.
* Initial Hox activity is set by transcription factors from early segmentation genes.
PcG Complexes and Their Functions
Function: PcG proteins are involved in maintaining the repression of genes that should not be expressed in particular cell types or at certain developmental stages. They achieve this through modifying chromatin structure, making it less accessible for transcription.
Complexes: The main PcG complexes involved are:
- Polycomb Repressive Complex 1 (PRC1)
- Polycomb Repressive Complex 2 (PRC2).
- Additional Complexes: PhoRC (less conserved, found in mammals and Drosophila) for DNA binding.
PRC1 and PRC2 are two biochemically distinct complexes, both evolutionarily widely conserved
PRC2: Methylates H3K27, a key modification for PcG-mediated repression.
PRC1: Recognizes H3K27me3 and monoubiquitylates H2AK118/119, which helps compact chromatin and inhibit transcription.
Components of the PcG complex are
continuously required to maintain
repression.
Mammals and plants have many PRC2 and PRC1 complexes –> Organism complexity, response to the environment
The concept of cellular memory
Remember:
* PcG proteins are required (only) for the maintenance of HOX repression rather than the position-specific establishment of HOX
activity.
* Establishment is done by the transcription factors encoded by early acting segmentation genes.
Components and function of PRC1
PC (Polycomb):
* Contains a chromodomain.
* Binds to H3K27me3 (a specific histone modification).
PSC (Posterior Sex Combs):
* Contains Zn finger and helix-turn-helix (HTH) domains.
SCE/dRing1 (Sex Combs Extra):
* Contains a RING Zn finger.
* Acts as a ubiquitin ligase, modifying H2A at K119ub.
PH (Polyhomeotic):
* Contains a Zn finger domain.
–> PRC1 (Polycomb Repressive Complex 1) is recruited to chromatin marked by H3K27me3.
Compacts chromatin to inhibit transcription.
* Prevents the SWI/SNF complex from remodeling chromatin.
* Prevent RNA Polymerase II Elongation:
* Stalls RNA Polymerase II, inhibiting transcription elongation.
Ubiquitylation:
* Monoubiquitylates histone H2A at K119.
* Contributes to chromatin compaction and transcriptional repression.
Note: the recruitment of PRC1 to chromatin can be a two-step process:
* H3K27me3-dependent recruitment: PRC1 is recruited to chromatin marked by H3K27me3.
* H3K27me3-independent recruitment: PRC1 can also be recruited independently of H3K27me3.
* PRC1 occupies large chromatin domains and provides stable repression of gene expression.
Components and function of PRC 2
E(z):
* Histone methyltransferase (HMT) that targets H3K27.
Known as enhancer of zeste.
Su(z)12:
* Zn finger protein.
* Acts as a cofactor for E(z), known as suppressor of zeste.
ESC/EED:
* Contains WD40 protein interaction domains.
* Determines the specificity of the HMT activity.
* Cofactor for E(z), known as extra sex combs.
P55:
* Also known as RbApAB46/48 or NURF55.
* Sometimes complexed with histone deacetylases (HDACs).
–> PRC2 Catalyzes the trimethylation of H3K27 (H3K27me3), a repressive chromatin mark.
- Recruitment of PRC1: H3K27me3 serves as a binding site for PRC1, linking PRC2 and PRC1 in gene silencing mechanisms. Establishes a repressive chromatin environment to maintain gene silencing.
PRC2 & PRC1 Cellular Memory:
They maintains the repressed state of genes through cell divisions, ensuring stable and heritable gene silencing.
PRC2 Functions in Different Organisms:
How is PcG silencing machinery recruited?
Histone Modifications: H3K27me3 by PRC2 attracts PRC1.
PREs and Transcription Factors: PREs and factors like PHO recruit PcG proteins. PhoRC binds PRE & can recruit PRC2 result: H3K27me
Noncoding RNAs: lncRNAs such as Hotair guide PcG complexes to target genes.
Protein Interactions: Direct interactions between PcG proteins and other chromatin proteins facilitate recruitment.
Chromatin State: Nucleosome depletion and histone modifications influence PcG binding.
- PhoRC can wrap DNA around itself
in presence of PRC1
PhoRC
Pho: DNA binding protein
SFMBT: can bind to certain methylated lysines on H3
PRE - PcG Response Element and how to define PREs (Schwartz and Pirrotta)
- First identified in bithorax complex in Drosophila
- Intergenic cis-acting DNA elements that PcG proteins bind to
- Loop back to promoter regions
- PREs are comprised of many transcription factor
binding sites (PHO and many others) - PRE have many bin ding sites for certain TFs
Definition of PREs - Schwartz and Pirrotta (2008)
(1) PREs attract H3K27me3
(2) they should form a new binding site for
PcG proteins when inserted at a new location
within the genome
(3) confers PcG-based repression to a
reporter gene.
Mechanism for PcG repression
- Generates large H3K27me domains. PRC2 initiates repression by adding H3K27me3 marks.
- PRC1 binds to H3K27me3 and ubiquitylates H2A
- PRC1 compacts chromatin, reducing accessibility.
- Prevents PolII elongation. PcG proteins block RNA Pol II and transcription factors.
- Form PcG bodies (subnuclear silencing
- compartments)
- Recruits HDACs, DNMTs (Repressors)
- Inhibits SWI/SNF ( ATPase remodeler) activity
How can we identify antagonists of PcG complexes?
Supressor screen in polycomb mutants
–> Identify mutations that suppress the phenotypic effects of PcG mutations, such as ectopic Hox gene expression.
- Use Drosophila or other model organisms with known PcG mutations.
- Screen for secondary mutations that restore normal phenotypes in these mutants. Look for wild-type looking flies
- Identify and characterize the genes responsible for the suppression. suppress ectopic HOX gene expression Identified trithorax group (TrxG) proteins
PcG and TrxG Interaction
Trithorax Group (TrxG) Proteins: act as antagonists to PcG, promoting gene activation. TrxG proteins modulate chromatin structure and affect gene regulation. They interact with transcriptional machinery to affect gene transcription and antagonize PcG
silencing.
TrxG proteins have many genomic targets.
The trxG of activators is a large and functionally diverse group of regulatory proteins. This may reflect the complexity of eukaryotic transcription.
Key Components:
* TRX (KMT H3K4me)
* ASH1 (KMT H3K4me, H3K36me)
* BRM, BRG1 = SWI/SNF complex (ATPase remodeler). Highly conserved and there are multiple forms of the complex.
Complex Balance:
The dynamic balance between PcG-mediated repression and TrxG-mediated activation regulates developmental genes and ensures proper gene expression patterns.
SET domain
originally identified as part of a larger conserved region present in the Drosophila Trithorax protein and was subsequently identified in Drosophila.
TrxG and SET domain proteins
* TRX (KMT H3K4me)
* ASH1 (KMT H3K4me, H3K36me)
* part of Compass complex
* Promotes H3K4me3, promotes transition of RNA Pol II to active elongation
H3K4-(me)3:
* TrxG binding
* Inhibits PcG binding
* inhibits HP1 binding
TrXG and Mediator
The Mediator complex is a multi-protein complex which is the interface of gene-specific activator proteins and formation of the preinitiation complex of RNA Pol II
It plays a crucial role in the regulation of gene expression by integrating signals from activators and repressors to modulate transcription.
PcG and TrxG bind the same element?
PRE
- Continuous inactivation required for PcG silencing
- Transcription through PRE causes loss of silencing
