Lecture 11: Chromatin & Transcription Regulation

Review the difference between heterochromatin and euchromatin and explain why transcription is most likely only in euchromatin. Include distinguishing facultative and constitutive heterochromatin

• Proteins to replicate and transcribe DNA must be able to recognize specific sequences of DNA

• How do they do this if histones and other compaction proteins are in the way?

• Chromatin is dynamic and can change states to allow proteins access to the DNA:

1. Heterochromatin-transcription is silenced because compaction is tight

2. Euchromatin-transcription is active because compaction is loose

• Some portions of some chromosomes will always be in the heterochromatin form:

1. Centromeres, Y chromosome

2. Called constitutive heterochromatin

• The chromatin state directly regulates transcription of genes. Position-effect Variegation (PEV) and Barr body formation

Explain the mechanisms (as we currently understand) that control position effect variegation (PEV) and X chromosome inactivation. Explain how these illustrate the role of heterochromatin in preventing gene expression

Position-Effect Variegation (PEV):

• PEV is the variable expression of a gene in a population of cells due to the gene’s location near highly compacted chromatin

• Example: Variable expression of the white gene in Drosophila eyes:

1. The wildtype, typical eye color of Drosophila is red and encoded by the wildtype version of the white gene (w+)

2. w+ is located on the X chromosome and usually resides in a region of euchromatin

3. If an inversion occurs, w+ is now located too close to heterochromatin

4. Cells that inherit the inversion cannot express w+

• PEV reflects states of facultative heterochromatin. Regions of chromosomes that are heterochromatic in some cells and euchromatic in others

• Heterochromatin spreads in a linear manner along the chromosome:

1. A second gene called roughest (rst+) is located near w+

2. rst+ creates a smooth surface on the eye cell

3. If heterochromatin spreads far enough to shut off rst+, cells will have a rough surface

X Chromosome Inactivation:

• Mammals will inactivate one of the X chromosomes in individuals that have more than one:

1. Inactive chromosomes are visible as Barr bodies

2. Method of dosage compensation between the biological sexes

• Inactivation of the chromosome is another example of facultative heterochromatin

• Inactivated chromosome is chosen at random in cells

• Very few genes can be expressed from the inactivated chromosome, now in a heterochromatic state

Expression of a long noncoding RNA (lncRNA) triggers formation of heterochromatin to inactivate the chromosome:

1. Called X inactive specific transcript (Xist)

2. Resides in nucleus permanently and is never translated

• Xist is transcribed from a region with the X chromosome called the X inactivation center (XIC)

• Stable transcription occurs only from chromosome that will be inactivated

• Xist RNA coats the X chromosome that produces it, which induces changes to chromatin state:

1. If XIC is moved to an autosome, that autosome will condense into a Barr body

2. If XIC is deleted from one X chromosome, the other will always become the Barr body

• Lingering questions:

1. Why is one chromosome chosen over the other?

2. Why does Xist associated only with the X chromosome that makes it?

3. How is inactivation maintained over mitotic division?

Define histone tail modification and chromatin remodeling as the primary methods of modifying chromatin states

• Specific modifications to histone proteins can affect the local chromatin structure

• The core histone proteins of the nucleosome have regions of linear amino acid sequence called tails. No secondary structures form

• Many of these amino acids have positive charges:

1. Primarily lysine (K) and arginine (R)

2. Any charge will make a molecule chemically reactive

Explain the mechanisms of histone tail acetylation and methylation

• Many different types of chemical modifications can be made to histone tails. A type of post-translational modification (PTM)

• Modifications to histone tails include (not limited to):

1. Methylation

2. Acetylation-always associated with opening chromatin

3. Phosphorylation

4. Ubiquitination

5. SUMOylation (addition of SUMO, a small ubiquitin-like molecule)

• These PTMs are carried out by specific enzymes:

1. Writers

2. Readers

3. Erasers

• Multiple combinations of modifications are possible:

1. One histone tail can have multiple amino acids modified

2. The same amino acid may be subject to different types of modifications

• Acetyl groups added to specific K residues will always open chromatin

• Methyl groups added to specific K residues or R residues will either open or close chromatin

• These modifications contribute to understanding how states of chromatin spread along chromosomes:

1. Ex. methylation at H3K9 serves as a binding site for heterochromatin specific proteins like HP-1

2. HP-1 can self-associate, which pulls adjacent nucleosomes together as well as promote H3K9 methylation on those nucleosomes

Define the histone code and explain how this alters chromatin states

• The pattern of modifications to histone tails that carries information to regulate DNA dependent events

• This pattern determines if other proteins can access DNA

Explain the mechanisms utilized by chromatin remodeling complexes to alter chromatin states

• A specific set of protein complexes use ATP to physically reposition nucleosomes or replace histones. Called chromatin remodeling complexes (ATP-dependent chromatin remodeling complexes)

• Nucleosome sliding: the energy of ATP hydrolysis will make the DNA less tightly associated with histones so it can be “pulled” out of the nucleosome

• Remodeling complexes can also replace all or some of the core nucleosome histones

• Several families of chromatin remodeling complexes

1. SWI/SNF (switch/sucrose-non-fermenting)

2. ISWI (imitation switch)

3. CHD (chromodomain-helicase-DNA binding)

4. INO80 (inositol requiring 80)

Describe why interactions between CREs, regulatory TFs, and the chromatin state affect transcription of genes

• As far as we know, gene transcription can only occur in regions of euchromatin. Therefore, there is a coordinated effort among CREs and regulatory TFs to alter heterochromatin or maintain euchromatin

• Often observe a cascading effect to open chromatin and bind the PIC for transcription to begin

• Insulators are the third type of CRE and function to organize chromatin:

1. Located between the enhancer/silencer and the gene core promoter

2. Limits the action of enhancers/silencers to the gene they should regulate

Define TAD and explain the connection between insulators and TAD formation. Recognize TADs as an example of chromatin organization

• Insulator sequences (in humans) bind a protein called CTCF (CCCCTC-binding factor) and form chromatin structures called topologically associating domains (TADs):

1. CTCF will interact with cohesion protein complex to form loops

2. Note these are different than the loops formed in mitotic chromosomes

• Once structured in a TAD, an enhancer/silencer must be within the same TAD to regulate a gene. TADs are dynamic structures!

• Mutations to insulators and/or regions that define the TAD boundaries can result in mutant phenotypes:

1. Ex: Polydactyly (specific subtype)

2. Genes IHH and EPHA4 are typically in separate TADs, so enhancer for EPHA4 will only affect this gene

2. Deletion of the boundary allows EPHA4 enhancer to activate transcription of IHH, a limb patterning gene

• Barrier elements are DNA sequences that appear to serve as binding sites for proteins that will open chromatin

• A mechanism to prevent heterochromatin from spreading too far along the chromosome. Another organizational strategy for maintaining the chromatin state

Recognize examples of experimental techniques that furthered understanding of transcriptional regulation

• Bioinformatics is a discipline of study that combines biology, computer science and information technology:

1. Computer programs are fed DNA sequence information for multiple types of analyses

2. Can predict DNA binding motifs, compare CREs across species, etc.

• The ENCODE project is attempting to map all CREs in the human genome:

1. Encyclopedia of DNA Elements

2. Expanded to include mapping of CREs in model organisms, called modENCODE

• Chromatin immunoprecipitation-sequencing (ChIP-seq) allows researchers to find all target genes of a particular TF within the entire genome of a cell type:

1. Chromatin is isolated and DNA is cross-linked to associated proteins

2. An antibody to TF of interest is added and will bind to matching TF

3. Precipitate DNA/TF complex and sequence DNA

• Chromatin conformation capture is the technique that identified TADs:

1. DNA and proteins in close proximity are cross-linked, fragmented, ligated and sequenced

2. Bioinformatics programs calculate how often DNA sequences are found within the same ligated molecule

Apply information to examples not presented in class

1. Position-Effect Variegation (PEV) & Facultative Heterochromatin

• Core Concept: A euchromatic gene placed adjacent to heterochromatin via chromosomal rearrangement can be stochastically silenced due to linear heterochromatin spreading.

• New Example (Human Cancer — CDKN2A / p16 Inversion):

  • In certain leukemia lines, a chromosomal translocation or inversion places the tumor suppressor gene CDKN2A (p16) adjacent to pericentromeric heterochromatin.

  • Application: Because heterochromatin spreads linearly without skipping, some clonal populations of cells silence p16 completely (leading to rapid cell division), while neighboring cells in the same tumor retain p16 expression in euchromatin. This produces intratumoral heterogeneity identical to the variegated red/white eye mosaicism observed with the white ($w^+$) and roughest ($rst^+$) loci in Drosophila.

2. X-Chromosome Inactivation & Xist lncRNA

• Core Concept: Xist lncRNA coats a single chromosome in cis, recruiting histone-modifying writers (e.g., HMTs) to form facultative heterochromatin (Barr body).

• New Example (Calico Coat Pattern in Cats & Human Fabry Disease):

  • Calico Cats: Female cats heterozygous for the X-linked coat color gene ($O$ = orange, $o$ = black) express Xist randomly in ~500–1000 cell stages, forming clonal patches of active paternal or maternal X chromosomes.

  • Fabry Disease (Humans): Females heterozygous for an X-linked mutation in the GLA gene show mosaic enzyme activity across cardiac and renal tissues. Cells that randomly inactivate the wild-type X chromosome display lysosomal storage dysfunction, whereas adjacent cells with the mutant X inactivated remain functional.

3. Histone Tail Modifications & The Histone Code

• Core Concept: Specific combinations of post-translational modifications (writers, erasers, readers) dictate chromatin openness (e.g., HATs add acetyl groups to open chromatin; HMTs modify residues to attract HP-1 or repressors).

• New Example (Oncogenic Histone H3K27M "Oncohistones" in Pediatric Glioma):

  • In diffuse intrinsic pontine glioma (DIPG), a single amino acid substitution occurs in histone H3 where Lysine 27 is mutated to Methionine (H3K27M).

  • Application: The H3K27M mutation acts as a potent inhibitor of the methyltransferase writer Polycomb Repressive Complex 2 (PRC2/EZH2). This prevents global trimethylation at H3K27 (a repressive mark), causing widespread loss of heterochromatin boundaries and inappropriate developmental gene expression.

4. Chromatin Remodeling & Regulatory Cascades

• Core Concept: Transcription factors recruit coactivators (HATs, kinases, ATP-dependent remodeling complexes like SWI/SNF) in a sequential cascade to slide or remove nucleosomes at core promoters.

• New Example (Steroid Hormone Response — Glucocorticoid Receptor Activation):

  1. Glucocorticoid hormone binds its cytosolic receptor, which translocates to the nucleus and binds a Glucocorticoid Response Element (GRE).

  2. The receptor recruits HAT coactivators (p300/CBP) to acetylate histone tails (e.g., H3K9, H4K8).

  3. Acetylation recruits ATP-dependent chromatin remodeling complexes (SWI/SNF) to slide nucleosomes away from the target gene's promoter.

  4. The nucleosome-free DNA allows binding of TFIID and the Pre-Initiation Complex (PIC) to initiate transcription.

5. TAD Boundaries, Insulators & Human Genetic Disease

• Core Concept: Insulator elements bound by CTCF and cohesin form Topologically Associating Domains (TADs) that restrict enhancers to target promoters within the same loop. Disruption of TAD boundaries allows ectopic enhancer-promoter cross-talk.

• New Example (Limb Malformations — WNT6 / IHH / EPHA4 Locus):

  • F-syndrome / Brachydactyly: Just like the IHH / EPHA4 polydactyly deletion shown in class, microdeletions or duplications of the CTCF-binding boundary between the WNT6 and IHH TADs remove the insulator.

  • Application: Without the TAD boundary, limb-specific enhancers that normally drive EPHA4 are hijacked to inappropriately activate WNT6 or IHH, disrupting digit patterning during embryonic development.

6. Experimental Techniques (ChIP-seq & Hi-C / 3C)

• Core Concept: ChIP-seq identifies specific genomic binding sites for transcription factors or histone marks; Chromatin Conformation Capture (3C/Hi-C) quantifies 3D interaction frequencies and maps TAD structures.

• New Example (Profiling Enhancer Landscape in Cardiac Hypertrophy):

  • ChIP-seq Application: Researchers isolate cardiac tissue, crosslink chromatin, and use antibodies against H3K27ac (an active enhancer mark) and GATA4 (a transcription factor) to map all active enhancers involved in heart disease.

  • Hi-C Application: By crosslinking, fragmenting, and ligating adjacent DNA loops, Hi-C interaction maps reveal how distal cardiac enhancers fold in 3D space to contact stress-response gene promoters.