Lecture 14: Heterochromatin

Characteristics of Heterochromatin

Hypermethylation of the DNA, hypoacetylation of histones, methylation of histone 3 at lysine 9 (H3K9me), and chromatin modifying proteins (ie. HP1 family members (scaffold for histone modifying proteins)).

GFP Fusion Array

Repeat GFPs in the heterochromatin/euchromatin region under the control of the alpha-globulin promoter in the RBCs. Use FACS to count cells in each line.

DNA Sequence Dependent Mechanism

Sequences of the DNA are recognized by binding proteins (HP1 type proteins) which bind and start the the heterochromatin formation.

Euchromatin

Uncondensed chromatin, associated with actively transcribed genes.

Position Effect Variegation (PEV)

When a gene that is normally expressed in euchromatin is caught up in a heterochromic region and is fully or partially silenced. Usually prevented by a barrier/insulator region. Place where heterochromatin is stopped varies by cells based on early embryo cells.

FACS

Cell counter which counts cells with certain levels of fluorescence.

Su(var)

Class of chromatin modifiers whose role is to extend heterochromatin. Mutation suppresses variegation.

E(var)

Class of chromatin modifiers whose role is to maintain euchromatin. Mutation enhances variegation.

Heterochromatin

Condensed chromatin, associated with gene silencing. A modification the makes genes resistant to expression and which is amenable to modification. (e.g. Barr body, telomeres, centromeres, areas around the nuclear envelope). 10% of the genome.

Facultative Heterochromatin

Heterochromatin associated with repressed genes that can be turned on or off during development.

RNAi-based Mechanism

Found in eucharyotic organisms (but not S. cerevisiae). Regions are transcribed as RNA and are recognized by the Argonaut protein. A large complex (RNA-induced transcriptional silencing complex (RITS)) forms which starts to interact with the modified histones and begins compacting the whole strand. Other proteins are recruited that increase the histone modifications and enhance compaction. An RNA-dependenet RNA polymerase binds and makes more siRNA which recruit more Argonaut which feeds forward for more modifications and condensation.

Gene Trap Construct

Introduce a antibiotic resistance marker gene into the middle of the gene. When the gene is expressed, the antibiotic resistance marker is expressed but the rest of the gene is not. Results in the expression of a fusion protein that contains a selectable marker and a truncated, non-functional version of the protein.

Constitutive Heterochromatin

More permanent form of heterochromatin which is associated with structural elements, repetitive sequences and transposons that are permanently silenced. Can be distinguished cytologically (e.g. Barr Body).