Molecular biology applied to biotechnology

ENHANCERS

NUCLEOSOME: THE MINIMAL STRUCTURAL UNIT OF

CHROMATIN

It is composed of 147 base pairs of DNA wrapped around an octamer formed by two of

each of the histones H3, H2A, H2B, and H4. The nucleosome serves as the physical

backbone of chromatin structure and as a layer of regulatory control of gene expression.

Tightly packed DNA is considered to be inactive, while unwrapped DNA is more

accessible for transcription. The genes in these regions are transcriptionally active.

Post-translational modifications (PTMs) on amino acids within the N-terminal tail of

histones regulate the physical properties of chromatin and its corresponding transcriptional

state (chromatin state). Most common PTMs: methylation, acetylation, phosphorylation,

ubiquitination.

Histone modifying enzymes: Writers, Erasers and Readers. Histone PTMs are catalyzed by

“writers” and “erasers” while their actions to govern DNA transcription are mediated by

“readers.”

Transcription requires a complex set of interactions. A simplified model for the main steps

of transcription initiation by RNA polymerase II (Pol II):

- Transcription activation starts with the binding of transcription factors on

enhancers

- Recruitment of chromatin modifiers or remodellers that alter chromatin structure

- Recruitment of other co-activators, e.g. Mediator complex, that act directly on the

assembly of basal transcriptional machinery, the so-called preinitiation complex

(PIC) 58

Within TADs, enhancer elements and active promoters form chromatin loops. Loops can

be mediated and/or stabilized by protein effectors, noncoding RNAs (ncRNAs), and

histone post translational modifications (PTMs). Chromatin loops within TADs organize

transcriptionally co-regulated genes and are important for defining cellular identity and

other physiological processes. Enhancers enable the establishment of spatiotemporal

patterns of gene expression during development.

ENHANCERS

The enhancer can be bound by activating transcriptional factors or by repressive

transcriptional factors, resulting in an activation or repression of transcription, respectively.

The loop formation, as we said, is mediated by cohesin sliding.

Enhancers, discovered in 1981, are classically defined as cis-acting DNA sequences that

increase the transcription of genes.

They generally function independently of orientation and at various distances from their

target promoter (or promoters).

Thousands of different enhancers are distributed in the mammalian genome, without

encoding any proteins, making their identification challenging. Many genes (ca 20,000) are

regulated by more than one enhancer. The clustering of genes poses a logistic problem for

enhancers to boost transcription from specific sets of neighboring genes during

development.

Promiscuous interplay between enhancers and promoters:

- one gene, multiple enhancers, one tissue

- one gene, multiple enhancers, more tissues

- gene competition for a shared enhancer → winner takes all 59

- gene competition for a shared enhancer → everybody wins

Chromatin features that define enhancers

The activity of enhancer regions is fundamentally determined by chromatin state and

binding of regulators:

- Chromatin accessibility

- Transcriptional factor (TF) binding motifs

- Post-translational modifications of histones

- Enhancer RNAs (eRNAs)

Chromatin as accessibility barrier

DNA at the active enhancer element is characterized by low nucleosome density and

hypersensitivity to DNase treatment. TF binding and DNA accessibility are highly

correlated. Not all accessible regions correspond to active enhancers. Open chromatin can

also be bound by repressive TFs.

Enhancers and regulator binding

Enhancers are not characterized by common general or coding DNA sequence. Enhancers

can contain short DNA recognition motifs (6-10 bp). Short DNA motifs act as binding

sites for sequence-specific transcription factors.

Pioneer TF → First TF to access the enhancer region leading to chromatin remodeling and

decompaction of nucleosomes. 60

Enhancer-associated histone modifications

Functional enhancers contain dynamic nucleosomes. H2A.Z and H3.3 increase the

dynamics and plasticity of nucleosomes at the enhancers. Outward movement of

H3K4me2-marked nucleosomes leads to enhancer activation.

H3K4me1 is the typical histone mark of enhancers. Active enhancers are also marked by

H3K27ac. H3K4me3 presence at the enhancer correlate with their activation status. Local

ratio of H3K4me1 to H3K4me3 serves as a more reliable indicator in enhancer prediction.

H3K27 is acetylated at flanking nucleosomes of active enhancers but methylated at poised

enhancers. Both classes show low nucleosomal density and hypersensitive to DNAse. Both

classes are bound by TFs, DNA binding active signaling effectors and coactivators. Poised

enhancers lack Pol II but can be occupied by the Polycomb repressive complex 2 (PRC2),

the “writer” of H3K27 methylation.

Enhancers can be primed for activation either at a later developmental time point or in

response to external stimuli and pre-labelled by H3K4me1 In closed chromatin, latent

enhancers are not pre-marked by known histone modifications à in the presence of external

stimuli the DNA becomes accessible, and flanking nucleosomes acquire H3K4me1 and

H3K27ac marks.

Enhancer-derived RNA (eRNA)

Enhancer-templated non-coding RNAs. Expression of eRNAs is positively correlated with

an enrichment of H3K27ac and depletion of H3K27me3. Dynamically regulated upon

signal-transduction. Reports of specific enhancer-derived transcription were first

documented in the locus control region (LCR) of the beta-globin gene clusters.

2d-eRNAs: Bidirectional eRNAs: 61

- Exhibit a 5’ cap but are not polyadenylated or spliced

- More common than 1D-eRNAs

- Highly specific for tissues and cell types

- Transcription is highly correlated with enhancer activity (e.g. preferentially enriched

at enhancers engaged in chromatin looping with promoters of protein-coding genes

and other enhancers)

- Highly correlated to expression of target PCGs

These regions are bound by lineage determining transcription factors (LDTFs) and

associated transcriptional co-regulators including subunits of Mediator, and the histone

acetyltransferases p300 and CBP. Activated enhancers produce eRNAs, which interact

with looping factors (e.g., cohesin complex) and facilitate/stabilize chromosomal looping.

eRNA mediates the loading of RNA Pol II at the promoter of the target gene.

CAGE identifies cell-type-specific enhancer usage → Enhancer activity can be detected

through the presence of balanced bidirectional capped transcripts.

Erroneous regulatory wiring between enhancers and target genes causes disease, you can

have:

- enhancer deletion

- disruption TF binding site

- insertion TF binding site

- enhancer duplication

- enhancer introduction

- promoter introduction

- promoter deletion

- enhancer hijacking

Polydactyly examples due to mutations into regulatory regions 62

SES: SUPER ENHANCERS

Super-enhancers (SEs) are putative enhancer clusters with unusually high levels of enhancer

activity. SEs are often in close proximity to critical cell identity-associated genes. SEs are

densely occupied by the master regulators and Mediator. A small set of lineage-defining SEs

determines cell identity in development and disease.

Median size an order of magnitude larger than that of normal enhancers (in mESCs 8,667

bp versus 703 bp).

SE vs TE: high RNA Pol II, eRNA, p300 and CBP

SE vs TE: high chromatin factors such as cohesin, mediators, chromatin remodelers

SE vs TE: high H3K27ac, H3K4me2 and H3K4me1

SE vs TE: increased chromatin accessibility as measured by DNase-seq

HIGHLY INTERCONNECTED ENHANCERS (HICE)

Highly interconnected enhancers (HICE) form 3D enhancer communities. In these hubs,

enhancers converge on transcriptional programs that define cell-identity. During

differentiation, connectivity is gained within lineage-specific HICE, and is lost in other cell

fates.

HICE are enriched in CTCF motifs and localized at (sub)TAD boundaries: - role in

genome architecture through CTCF recruitment and cohesin-mediated loop extrusion.

HICE are enriched for binding of TFs, cofactors and mediators:

- correlation of TF occupancy at interacting enhancers (CIST) 63

- role in nuclear condensates formation through phase separation (TFs and

coactivators form liquid phases that act to compartmentalize and concentrate

regulatory machineries)

ATAC-seq: Assay for Transposase-Accessible Chromatin using sequencing

ATAC-seq relies on next generation sequencing (NGS) library construction using the

hyperactive transposase Tn5. Engineered Tn5 enzyme that inserts adapters instead of a

transposon. NGS adapters are loaded onto the transposase, which allows simultaneous

fragmentation of chromatin and adapter integration into open chromatin regions

(tagmentation).

Main advantages over other open chromatin assays:

- Simple library preparation protocol, that can be completed in under 3 hours

(compatible with clinical timescales)

- No sonication or phenol-chloroform extraction / No antibodies / No enzymatic

digestion

- Low starting material (500 to 50K cells)

ATAC-seq reads are mapped to the genome to create a signal of Tn5 insertion events. The

depletion of ATAC-seq signal defines TF binding, referred as footprint. Bioinformatic

approaches predict TF binding based on the sequencing motifs present in the footprint.

ENHANCERS EVALUATION: EXPERIMENTAL APPROACHES

Single histone modifications are associated with activation or repression. The combinatorial

profile of HMs provides a more accurate definition of chromatin states. 64

REPETITIVE DNA SEQUENCES

Genomics studies organization, function and evolution of DNA sequences which are

contained in a specific genome.

The Human Genome contains 3,1x109 base pairs (bp), packed into 23 couples of linear

molecules, named chromosomes

- smallest chromosomes: ~500.000 bp

- largest chromosomes: ~250.000.000 bp

Bacterial genome dimension correlates with number of genes.

DNA CONTENT(C) = The sum of all the sequences which composes a genome

COMPLEXITY (S) = The sum of all the different sequences which composes a genome

Organisms with similar complexity (s) can contain different quantities of the same

sequences

Eukaryotic genome size fails to correlate well with apparent complexity →“C-value

paradox”:

- Only a (small) part of the genome is coding for protein information

- Some sequences could be represented in multiple copies, yet quantity does not

correlate with their complexity

In the human genome, < 2% encodes genes (exons). Humans share > 85% of genes with

mice. Humans share > 98% of genes with chimpanzees. Evolution thus relies largely on

changes in non-coding regions of genomes.

Only 25% is gene sequence, the 75% is extra genes sequences:

- 50% repetitive elements